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Studies on histone modification and chromatin structure in developing trout testi Honda, Barry M. 1975

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STUDIES ON HISTONE MODIFICATION AND CHROMATIN STRUCTURE IN DEVELOPING TROUT T E S T I S  by  BARRY M. HONDA B.Sc,  McMaster U n i v e r s i t y ,  1971  THESIS SUBMITTED IN PARTIAL FULFILLMENT THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the  Department o f B i o c h e m i s t r y Faculty  We  accept  this  to  the required  of Medicine  thesis  as  conforming  srta*id/ard  'S'ep-hember 1975 University  of B r i t i s h  Columbia  In presenting this thesis  in partial fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this  thesis  for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  "Bl^CM 6 M  KTfcM  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  Sep*  /9 ,  /f?S  ABSTRACT During  spermatogenesis  i n rainbow t r o u t , a synchronous  development o f stem c e l l s occurs, with  •> s p e r m a t o c y t e s -*• s p e r m a t i d s replacement of the histones  -*• m a t u r e  sperm  i n chromatin  by  protamines. PART A:  Histone  Along  with  methylation histone  ation of specific observed. the  residues of histones  This histone methylation  large d i p l o i d  actively  lysyl  a c e t y l a t i o n and p h o s p h o r y l a t i o n ,  stem c e l l s  synthesize  methylation  occurs  and p r i m a r y  DNA and h i s t o n e s .  p l a c e m e n t o f h i s t o n e s by p r o t a m i n e . ments s u g g e s t the c e l l  deacetylation for to  occurring after  o f h i s t o n e H4.  histone phosphorylation cell  spermatocytes,  Other  which histone  i n the r e -  labelling i s a late  in  experievent i n  t h e s y n t h e s i s , a c e t y l a t i o n and  This methylation or chromatin  may be  condensation  necessary prior  division.  PART B;  Chromatin subunit  structure  When a sample o f t r o u t t e s t i s micrococcal discrete tiples  predominantly  h a s no r o l e  t h a t h i s t o n e H4 m e t h y l a t i o n  cycle,  H3 and H4 c a n be  In spermatids,  i s m i n i m a l and s o p r o b a b l y  methyl-  nuclease,  i s cleaved  fragments approximately  thereof.  isolated  t h e DNA  The same DNA  chromatin,  nuclei  i s digested  with  almost e n t i r e l y t o  200 b a s e p a i r s l o n g and m u l -  f r a g m e n t s c a n be o b t a i n e d  as opposed t o i n t a c t  nuclei,  when  i s nuclease  -it-  digested. chromatin  T h e s e DNA f r a g m e n t s c a n a l s o be f o u n d isolated  from n u c l e a s e - d i g e s t e d  nuclei.  Sedimentation through sucrose  gradients, or velocity  sedimen-  tation  "subunits"  i ndiscrete  i n an a n a l y t i c a l  atin  subunits  into lis  etc.  species.  Subunits  2B  u l t r a e e n t r i f u g e separates (monomer), 16S ( d i m e r ) ,  NaCl) o r d i v a l e n t c a t i o n s  mately  subunits  1.2 a n d c o n t a i n  specific histone  histone  H6.  22S  chrom-  (trimer)  c a n a l s o be f r a c t i o n a t e d o n a S e p h a r o s e  column e q u i l i b r a t e d and r u n i n low s a l t .  Chromatin  these  (-5 mM)  cause  High s a l t  subunit  precipitation.  have a protein:DNA r a t i o a l l the histones,  of approxi-  including the trout-  T h e r e a r e however no d e t e c t a b l e  chromosomal p r o t e i n s .  Mg  + +  (>40 mM  non-  p r e c i p i t a t e s o f t h e 11S  c h r o m a t i n monomers, when p e l l e t e d , a r e t h i n  and c l e a r ,  while  oligomer  This  reflect  Mg  pellets  a more s y m m e t r i c a l are  deficient  or ordered  i n histone  larger oligomers,  packing  This histone  may c r o s s l i n k t h e  i n a disordered  are consistent with  histones.  Mg  + +  complex.  the subunit  These s u b u n i t s  n u c l e a s e - s e n s i t i v e DNA s p a c e r  could  o f 11S monomers, w h i c h  s t r u c t u r e , b a s e d o n 200 b a s e p a i r l o n g  DNA a s s o c i a t e d w i t h by  Hi.  resulting  These r e s u l t s chromatin  a r e t h i c k and w h i t e .  regions,  model o f  regions o f  w o u l d be  separated  and c r o s s l i n k e d by  histone H i . Testis (meiotic  c o n s i s t i n g predominantly o f e a r l y  tissue,  containing mainly  spermatids  nucleohistone)  s i m i l a r y i e l d s o f DNA f r a g m e n t s a n d 11S s u b u n i t s . testis  (protamine has r e p l a c e d  the histones)  gives Later  stage  h o w e v e r , g i v e s no  DNA  fragments or  differences histone.  11S  subunits.  This  presumably r e f l e c t s  i n s t r u c t u r e between n u c l e o p r o t a m i n e and  large  nucleo-  -iv-  TABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS LIST OF TABLES  ' iv viii  LIST OF FIGURES  i%  ACKNOWLEDGMENT  xii  PART A;  HISTONE METHYLATION  INTRODUCTION  1  I.  1  II.  III. IV.  The H i s t o n e s H i s t o n e I n t e r a c t i o n s w i t h DNA and Other Histones  7  Covalent M o d i f i c a t i o n s o f the Histones  9  Spermatogenesis  MATERIALS AND METHODS I. II.  C h e m i c a l s and A b b r e v i a t i o n s I s o l a t i o n and C h a r a c t e r i z a t i o n o f i n v i v o [ ^C]methylated Trout T e s t i s Histones (a) [ C] m e t h y l l a b e l l i n g o f h i s t o n e s (b) S t a r c h g e l e l e c t r o p h o r e s i s (c) D e t e r m i n a t i o n o f m e t h y l a t e d amino a c i d s .. (d) D e t e r m i n a t i o n o f c e l l u l a r L - m e t h i o n i n e and S - a d e n o s y l - L - m e t h i o n i n e S i t e s of i n v i v o Methylation o f Trout T e s t i s Histones (a) Automated p r o t e i n s e q u e n c i n g (b) [ *C)methyl p e p t i d e s o f h i s t o n e H3 (c) T r y p t i c p e p t i d e s o f [ ^ C l m e t h y l l a b e l l e d h i s t o n e H4 (d) Sequence d e t e r m i n a t i o n s on [ ^ C ] m e t h y l l a b e l l e d t r y p t i c peptides 1  Ilf  III.  1  ll  15 18 18 19 19 20 20 21 22 22 22 23 24  -v-  Page  IV.  V.  Histone Methylation i n D i f f e r e n t C e l l Types from D e v e l o p i n g T r o u t T e s t i s (a) C e l l s e p a r a t i o n s (b) S t a r c h g e l e l e c t r o p h o r e s i s f o r h i s t o n e s .. (c) P u l s e - c h a s e t u r n o v e r e x p e r i m e n t s ......... (d) R a d i o a c t i v i t y a n a l y s i s K i n e t i c s o f M e t h y l L a b e l l i n g o f H i s t o n e H4 .... (a) P r e p a r a t i o n o f [ *C]methyl l a b e l l e d h i s t o n e H4 (b) R a d i o a c t i v i t y i n t h e m o d i f i e d s p e c i e s o f h i s t o n e H4  25 25 26 26 27 27  ll  RESULTS I.  II.  III.  IV.  27 28 29  I s o l a t i o n and C h a r a c t e r i z a t i o n o f i n v i v o Methylated Trout T e s t i s Histones (a) Which h i s t o n e s a r e m e t h y l a t e d ? (b) Which m e t h y l a t e d b a s i c amino a c i d s a r e present? Sites of i n vivo Methylation of Lysyl R e s i d u e s Tn T e s t i s H i s t o n e s (a) H i s t o n e H3 (b) H i s t o n e H2B (c) H i s t o n e H6 (d) H i s t o n e H4 (a) H i s t o n e M e t h y l a t i o n i n t h e D i f f e r e n t C e l l Types from D e v e l o p i n g T r o u t T e s t i s .. (b) Turnover o f m e t h y l groups on h i s t o n e l y s y l residues ^ K i n e t i c s o f H i s t o n e H4 M e t h y l a t i o n  DISCUSSION  29 30 30 37 37 45 45 47 51 56 56 64  Methylation Sites  64  H i s t o n e M e t h y l a t i o n i n D i f f e r e n t C e l l Types  66  T u r n o v e r o f H i s t o n e M e t h y l Groups  66  K i n e t i c s o f H i s t o n e H4 M e t h y l a t i o n  67  PART B:  CHROMATIN SUBUNIT STRUCTURE  INTRODUCTION  70  -vi-  Page MATERIALS AND METHODS I. II. III. IV. V.  VI. VII. VIII.  . ....  C h e m i c a l s and A b b r e v i a t i o n s  75  P r e p a r a t i o n and N u c l e a s e D i g e s t i o n o f N u c l e i o r I s o l a t e d Chromatin  75  D e t e r m i n a t i o n o f Developmental S t a g e s (histone:protamine r a t i o ) of Testes  76  P r e p a r a t i o n o f C h r o m a t i n S u b u n i t s from D i gested N u c l e i  76  A n a l y s i s o f DNA Fragments Produced by N u c l e a s e Digestion (a) I s o l a t i o n o f DNA fragments (b) Q u a n t i t a t i o n o f DNA (c) N o n - d e n a t u r i n g 2.5% p o l y a c r y a l m i d e g e l e l e c t r o p h o r e s i s o f DNA (d) D e n a t u r i n g 9 9% formamide, 6% p o l y a c r y l a mide g e l s o f DNA  79  Sepharose 2B Chromatography o f C h r o m a t i n Subunits  80  V e l o c i t y S e d i m e n t a t i o n E x p e r i m e n t s on Subunits  80  P r o t e i n Composition o f Chromatin Subunits (a) I s o l a t i o n o f s u b u n i t s from n u c l e i o r whole c h r o m a t i n (b) G e l e l e c t r o p h o r e s i s o f p r o t e i n s (c) Q u a n t i t a t i o n o f p r o t e i n s  RESULTS I.  II.  75  77 77 78 78  82 82 83 84 86  C h a r a c t e r i z a t i o n o f DNA by M i c r o c o c c a l N u c l e a s e (a) DNA fragments from (b) DNA fragments from  Fragments Produced Digestion digested nuclei d i g e s t e d c h r o m a t i n ....  86 86 92  P r e p a r a t i o n o f I s o l a t e d C h r o m a t i n S u b u n i t s .... 95 (a) S u c r o s e g r a d i e n t p r e p a r a t i o n o f chromatin subunits 95 (b) S e d i m e n t a t i o n v e l o c i t y a n a l y s i s o f chromatin subunits 97 (c) Sepharose 2B chromatography 101  Page III.  IV. V.  Trout T e s t i s Chromatin Subunit S t r u c t u r e : Comparison o f N u c l e o h i s t o n e and N u c l e o protamine  101  P r o t e i n Composition  103  The B e h a v i o u r  o f Chromatin  o f Chromatin  Subunits  Subunits  i n Salt  DISCUSSION DNA  110  Digestion Patterns  P r o t e i n Composition Subunit P a r t i c l e s Chromatin  ....  110  and A r r a n g e m e n t i n C h r o m a t i n  Structure During T e s t i s Maturation  Conclusion BIBLIOGRAPHY  ... 107  112 H8 119 123  -viii-  L I S T OF TABLES Page PART A: I. II. III.  Nomenclature and p r o p e r t i e s o f h i s t o n e s  3  Sequence d a t a  4  II.  available  [ *C]methyl l a b e l l e d h i s t o n e H3 1!  PART B; I.  HISTONE METHYLATION  CHROMATIN SUBUNIT  f o r the histones  peptides  derived  from 44  STRUCTURE  Chromatin Subunits-Sedimentation (a) Scanner O p t i c s (b) Schlieren Optics M i c r o c o c c a l N u c l e a s e D i g e s t i o n on and N u c l e o p r o t a m i n e  Velocity  Data 98 99  Nucleohistone 104  -ix-  LIST OF FIGURES PART A;  HISTONE METHYLATION  Figure  Page  1.  L i C l g r a d i e n t s e p a r a t i o n o f [ "*C] m e t h y l l a b e l l e d histones  31  2.  S e p a r a t i o n o f [ *C]methyl h i s t o n e s from p r o t a m i n e s .  32  3.  Preparation of [^C]methyl l a b e l l e d histone t i o n s on B i o - G e l P-10  33  4.  P r e s e n c e o f [ ^ C ] m e t h i o n i n e and [ ^ C l m e t h y l amino acids i n the histones  35  5.  S e p a r a t i o n o f e - N - m e t h y l - l y s i n e s on a T e c h n i c o n amino a c i d a n a l y s e r  36  Automated d e g r a d a t i o n t e s t i s h i s t o n e H3  39  6.  1  JI  frac-  1  of [ ^C]methyl l a b e l l e d 1  7.  Recovery o f a l a n i n e d u r i n g automated s e q u e n t i a l d e g r a d a t i o n o f h i s t o n e H3  40  8.  Sephadex G-50 chromatography o f N - b r o m o s u c c i n i m i d e d e r i v e d p e p t i d e s o f [ C ] m e t h y l h i s t o n e H3  42  9.  Sephadex G-10 chromatography o f H3 t r y p t i c tides  43  10.  Automated sequence a n a l y s i s o f [ C ] m e t h y l l a b e l l e d t e s t i s h i s t o n e H2B  46  11.  Sephadex G-25 chromatography o f t r y p t i c o f m a l e y l a t e d [ *C]methyl h i s t o n e H4  48  pep-  1 4  peptides  ll  12.  Paper chromatography o f t r y p t i c p e p t i d e s o f m a l e y l a t e d H4  49  13.  Sequence a n a l y s i s o f [ ^ C ] m e t h y l h i s t o n e H4  50  14.  The s i t e s o f a c e t y l a t i o n and m e t h y l a t i o n o f t e s t i s h i s t o n e s H3 and H4  52  I n c o r p o r a t i o n o f L - [ m e t h y l - H ] m e t h i o n i n e and L - [ ^ C ] l y s i n e i n t o d i f f e r e n t c e l l t y p e s from trout testis  54  15.  3  1  -x-  Figure 16.  17. 18.. 19.  20.  21.  Page S y n t h e s i s and M e t h y l a t i o n o f h i s t o n e s i n t h e d i f f e r e n t c e l l types o f t r o u t t e s t i s  55  Turnover o f [ histones  57  1I+  C] methyl groups  Incorporation of [ H]arginine into with time-cell v i a b i l i t y 3  2.  3. 4.  5.  6.  7.  8.  testis  testis  nuclei 58  The a c e t y l a t e d a n d p h o s p h o r y l a t e d s p e c i e s o f h i s t o n e H4, s e p a r a t e d by s t a r c h g e l e l e c t r o phoresis  60  [ H ] l y s i n e i n c o r p o r a t i o n i n t o h i s t o n e H4 w i t h t i m e , a s d e s c r i b e d by L o u i e a n d D i x o n (84)  61  [^C]methyl incorporation into f u n c t i o n of time  63  3  PART B: CHROMATIN SUBUNIT 1.  i n trout  h i s t o n e H4 a s a  STRUCTURE  S c a n s o f DNA f r o m h i s t o n e and p r o t a m i n e s e p a r a t e d on 2.5% p o l y a c r y l a m i d e g e l s  stage  nuclei 88  P l o t o f DNA band number v e r s u s s q u a r e r o o t o f band m o b i l i t y f o r DNA s e p a r a t e d on 2.5% p o l y a c r y l amide g e l s  89  D e n a t u r i n g 99% f o r m a m i d e , 6% p o l y a c r y l a m i d e g e l s e p a r a t i o n o f DNA f r a g m e n t s  90  C a l i b r a t i o n of mobility versus single stranded l e n g t h f o r n u c l e i c a c i d s s e p a r a t e d on f o r m a m i d e gels  91  C h a r a c t e r i z a t i o n o f DNA f r a g m e n t s f r o m n u c l e i and i s o l a t e d c h r o m a t i n  93  intact  2.5% p o l y a c r y l a m i d e g e l s e p a r a t i o n o f DNA e x t r a c t e d from c h r o m a t i n s u b u n i t m o n o m e r s — m i n o r bands  94  C h a r a c t e r i z a t i o n o f DNA i n c h r o m a t i n across a sucrose gradient  96  Sedimentation v e l o c i t y digested nuclei  analysis  subunits  of chromatin  from 100  -xi-  Figure 9.  10. 11.  Page S e p h a r o s e 2B c h r o m a t o g r a p h y o f c h r o m a t i n l a t e d from n u c l e a s e - d i g e s t e d n u c l e i Histone sucrose  content i n chromatin gradient  P r e c i p i t a t i o n of chromatin and d i v a l e n t c a t i o n s  subunits  subunits  iso-  across  102 a 106  by  monovalent 108  -xii-  ACKNOWLEDGMENT I w i s h t o thank Dr. Gordon H. Dixon f o r h i s i n i t i a l guidance and s u p p o r t d u r i n g t h e s e s t u d i e s .  A l s o , those  f a c u l t y members who a t one time o r a n o t h e r s e r v e d on my a d v i s o r y committee — Drs. P.D. Bragg, M i c h a e l Smith, Gordon Tener, S.H. Zbarsky — gave me g r e a t h e l p and s u p e r vision.  I owe a g r e a t debt t o my p r e s e n t a d v i s o r , Dr.  P e t e r Candido, f o r h i s comments, encouragement, e n t h u s i a s t i c i n t e r e s t and f r i e n d s h i p d u r i n g t h e c o u r s e o f t h i s work. I a l s o w i s h t o thank t h e many p e o p l e i n t h e department who gave me a d v i c e and h e l p , e s p e c i a l l y Drs. S t e w a r t G i l m o u r , Andrew L o u i e , I a n G i l l a m , S h i r l e y G i l l a m , and my c o l l a b o r a t o r and i n s p i r a t i o n f o r much o f t h e l a t e r work, D r . David B a i l l i e .  J o e Durgo r e q u i r e s s p e c i a l mention f o r  singlehandedly keeping the l a b o r a t o r y instruments working f o r us. L a s t l y , I s h o u l d acknowledge t h e f o u r y e a r s o f s u p p o r t from t h e N a t i o n a l Research C o u n c i l o f Canada, from whom I r e c e i v e d a C e n t e n n i a l S c i e n c e S c h o l a r s h i p .  PART A:  HISTONE IffiTHYLATION -1-  INTRODUCTION I.  the  The  Histones  The  histones  are basic proteins associated with  chromosomes o f e u k a r y o t i c 1-4).  refs.  described  Miescher  organisms  and l a t e r  it  techniques  and  (5)  first  i s o l a t e d and  identifiable  fractionation  t h a t t h e r e a r e f i v e major b y amino a c i d  species of  composition,  sequence,  proteins,  present  generally  r e m o v e d f r o m t h e DNA b y e x t r a c t i o n w i t h  solution.  cribed  i n approximately  Recently  from Neurospora c r a s s a  found  i n yeast,  could  account  contrary  to earlier  chromatin  er  eukaryotes. A given  (6);  result  reports  (6).  (8),  t y p e may  i n charge  acid side chains. birds, (4),  fish,  DNA, a r e  acid or were  des-  but p r o t e o l y s i s  Hence i t i s l i k e l y  the nuclear  structure i n fungi are similar  histone  These  H i a n d H3 have n o t b e e n  that,  basic proteins t o those  show some h e t e r o g e n e i t y  the p r e s e n c e o f m i n o r components w h i c h v a r y to changes  types  S a c c h a r o m y c e s c e r e v i s i a e (7),  for this  and  equal weight with  t h e same 5 h i s t o n e  With  and a n a l y s i s  e l e c t r o p h o r e t i c and c h r o m a t o g r a p h i c p r o p e r t i e s .  salt  see  apparent heterogeneity.  f o r histone  i s now r e c o g n i z e d  histone  (for reviews,  the histones, but f u r t h e r c h a r a c t e r i z a t i o n o f these  p r o t e i n s was hampered b y t h e i r improved  Kossel  DNA i n  of high-  due t o  i n sequence, o r  f o l l o w i n g c o v a l e n t m o d i f i c a t i o n o f amino  In a d d i t i o n , nucleated  amphibia, r e p t i l e s )  erythrocytes ( i n  c o n t a i n a s p e c i a l h i s t o n e , H5  and r a i n b o w t r o u t t i s s u e s h a v e an e x t r a h i s t o n e  H6  (9).  - 2 -  The  sperm c e l l s  basic  o f many a n i m a l  species  " p r o t a m i n e s " w h i c h may r e p l a c e  contain  small  thehistones  highly  i n chromatin  (10). Table the  I presents  histones  will  from c a l f  thymus a n d o t h e r  Table  1974  i n London,  I I summarizes t h e s o u r c e s  a v a i l a b l e f o r the histones.  o n l y H i has n o t been c o m p l e t e l y halves  o f H i f r o m two d i f f e r e n t  Noteworthy ing  portions of servation  o f amino a c i d  sequenced; however, have been  sequence a c r o s s  H3  a n d H4 h a v e b e e n f o u n d  H4  has a phosphorylated  similar  di-  to specific  elution lysyl  associated  residues.  as tetramers  NH2-terminal s e r i n e  (37).  Histone  tree.  cluster-  f r o m DNA, a n d e x t e n -  (35)  More r e c e n t l y , (32-34) . and c o n t a i n s  (except  (12)  i n H4 h a s  H3 c o n t a i n s  and sometimes t r i m e t h y l - l y s i n e a t r e s i d u e s  f o r pea  and p o s i t i o n s  The presence o f 3 - p h o s p h o h i s t i d i n e  been r e p o r t e d  con-  i n terms o f a r g i n i n e NH2-terminal  a n d e - N - a c e t y l - l y s i n e a t p o s i t i o n 16  recently  sequenced.  are (i)the cluster-  e-N-mono- a n d d i m e t h y l - l y s i n e a t p o s i t i o n 20  (36).  adjoining  a phylogenetic  extreme sequence c o n s e r v a t i o n ,  e-N-methylation o f s p e c i f i c  5,8,12  species,  h i s t o n e molecule and ( i i ) t h e s t r i k i n g  i n g o f b a s i c amino a c i d s , s a l t  H4)  sequence  Of t h e f i v e m a j o r  species  H3 a n d H4 a r e v e r y  thesis  a t t h e CIBA  (and c o v a l e n t m o d i f i c a t i o n s )  o f amino a c i d  Histones content,  a  This  (11).  f e a t u r e s o f t h e sequence d a t a  o f basic residues  sive  sources.  u s e t h e H i t o H6 n o m e n c l a t u r e p r o p o s e d  Foundation meetings  data  t h e n o m e n c l a t u r e a n d some p r o p e r t i e s o f  e-N-mono-/  9,27  (17-20)  -3-  TABLE I  Histone HI  A l t e r n a t e Nomenclatures I,  F l , KAP,  H2A  Ilbi,  H2B  lib*,  H3  III,  H4  IV,  H5 H6  lysine-rich  F 2 a 2 , LAK) s l i g h t l y '' l y s i n e F 2 b , KAS ) r i c h  F 3 , ARE  ^  arginine  F 2 a l , GRK  J rich  V, F 2 c , KSA, e r y t h r o c y t e specific T, AKP,  trout-specific  Molecular  Weight  NH -terminus 2  21,000  Acetyl-Ser  14,000  Acety'l-Ser  13,800  Pro  15,300  Ala  11,300  Acetyl-Ser  %20,000  Thr  ^14,500  Pro  -4-  TABLE I I  Histone H4  Source* calf  thymus  pig pea rat H3  calf  thymus  mollusc,cycad, shark, sea u r c h i n trout testis calf  thymus  trout H2A  calf  testis thymus  trout  HI  complete complete  testis  16  complete complete  17 18  complete  19  complete residues 1-40  20 21  residues  1-25  22  complete residues 1-22  23 22  complete  24  p a r t i a l (missing 2 i n t e r n a l sequences)  r a b b i t thymus c a l f thymus  r e s i d u e s 1 - 107 residues 1-72  trout  r e s i d u e s 108 - 210  testis  H5  chicken  H6  trout  erythrocyte  testis  Reference 12,13 14 15  complete complete  chicken pea carp  H2B  E x t e n t o f Sequence  25  26,27 26 28  residues 1-70 25 i n t e r n a l  29,30  r e s i d u e s 1 - 29  31  * P a r t i a l sequences from a few o t h e r s o u r c e s , t e s t i s , a r e a v a i l a b l e f o r H4 (see r e f . 1 ) .  including  trout  -5-  and  t o a l e s s e r e x t e n t a t r e s i d u e s 4 (21)  and  36  (13,21);  £-N-acetyl-lysine has been l o c a l i z e d a t r e s i d u e s 14,23 and  l e s s a t r e s i d u e s 9,18  t i o n may  (22).  Low  l e v e l s of H3  o c c u r a t s e r y l r e s i d u e s 10 and  28  e s t i n g t h a t sequence m i c r o h e t e r o g e n e i t y f o r pea  h i s t o n e H3,  t a i n a l a n i n e and a l s o occur  f o r h i s t o n e s HI  (26)  and  examples o f t h i s m i c r o h e t e r o g e n e i t y is s t i l l  unresolved  H i s t o n e s H2A  (e.g. r e f s .  and H2B  H5  60%  two  (47) .  can o c c u r  Whether more i n the  19,39,48).  amino a c i d sequences. (33,34).  H4  a t s e r i n e 6 (35) and  18  and  these  T r o u t t e a t i s H2A  i s phosphorylated  has  and i s  and a c e t y l a t e d (to low  levels)  i s a c e t y l a t e d a t l y s y l r e s i d u e s 5,10,13  (22) .  H i s t o n e HI d i f f e r s in  less  Dimers between  a t the NH2-terminal s e r i n e (35) T e s t i s H2B  in dissociating  i n having  an NH2-terminal sequence homologous t o t h a t o f H4,  a t l y s i n e 5 (40).  histones  a l s o have c l u s t e r i n g o f b a s i c amino  h i s t o n e s have been r e p o r t e d  phosphorylated  con-  M u l t i p l e components  a t a lower s a l t c o n c e n t r a t i o n and  h i g h l y conserved  It i s i n t e r -  o f molecules  a c i d s a t the NH 2 - t e r m i n u s , but d i f f e r from H3, from DNA  phosphoryla-  has been r e p o r t e d  where a t r e s i d u e 96,  40% have s e r i n e (19).  (38).  (17)  from the o t h e r major h i s t o n e  i t s larger size, stoichiometry  other histone  species  [ h a l f the amount o f  (41)], heterogeneity w i t h i n a given t i s s u e  charge d i s t r i b u t i o n  (NH2  and  carboxyl-terminal regions  any (26),  are  b a s i c , middle i s h y d r o p h o b i c ) , d i s s o c i a t i o n from DNA a t low c o n c e n t r a t i o n s , and  i t s increased evolutionary v a r i a b i l i t y ,  salt  -6-  seen v i a e l e c t r o p h o r e t i c s t u d i e s ( 4 2 ) . sequence r e p o r t e d f o r t r o u t t e s t i s  The  carboxyl-terminal  (28) i s i n t e r e s t i n g i n  t h a t a l a r g e number o f r e p e a t i n g sequences can be found — 3 t e t r a p e p t i d e sequences - L y s - S e r - P r o - L y s - , phosphorylated,  and  (i)  each p o t e n t i a l l y  ( i i ) s i x h e x a p e p t i d e sequences a l l de-  r i v a b l e from an a r c h e t y p a l sequence T h i s r e s u l t s t r o n g l y suggests  -Val-Ala-Ala-Lys-Lys-Pro-.  t h a t l a r g e portions of  the  m o l e c u l e a r o s e t h r o u g h r e p e a t e d p a r t i a l gene d u p l i c a t i o n ( 2 8 ) . H i has been r e p o r t e d t o c o n t a i n e - N - p h o s p h o l y s i n e ( 3 7 ) .  How-  e v e r , a s i d e from t h e N H 2 - t e r m i n a l a c e t y l - s e r i n e , no a c e t y l a t i o n o r m e t h y l a t i o n o f t h i s h i s t o n e has been o b s e r v e d , e x c e p t perhaps f o r t r a c e amounts o f N ^ - m e t h y l - h i s t i d i n e H i s t o n e H6  i s present  tone) i n t r o u t t i s s u e s ( 9 ) . a b i l i t y from c h r o m a t i n l i k e H2B  and  (43).  i n low amounts (1% o f t o t a l  his-  I t i s s i m i l a r to Hi i n e l u t l y s i n e , a l a n i n e c o n t e n t , but i s  i n s i z e and l y s i n e r a r g i n i n e r a t i o .  There i s as  yet  no known r o l e f o r t h i s h i s t o n e i n t r o u t t i s s u e s .  I t i s now  g e n e r a l l y b e l i e v e d t h a t the h i s t o n e s are  t h e s i z e d d u r i n g S phase, on s m a l l p o l y r i b o s o m e s plasm (44-46).  The  i n the c y t o -  i s o l a t i o n o f h i s t o n e mRNA t r a n s c r i p t s  w h i c h l i k e l y do not c o n t a i n 3' p o l y A demonstration  syn-  (49)  (50) has l e d t o the  o f m u l t i p l e (500-1000) c o p i e s o f the  histone  genes (51,52) i n sea u r c h i n , t h e l o c a l i z a t i o n o f h i s t o n e genes by i n s i t u h y b r i d i z a t i o n t o D r o s o p h i l a m e l a n o g a s t e r  polytene  -7-  chromosomes in  (53) , and  closed circular  translation  the  recent  plasmid  of histones  DNA  not  resolved  synthesis, bind  (58),  (see r e f . 28,  histones  t o DNA.  (54).  are  half-lives  o f DNA  histones  the  (57),  t h i s problem i s  to the  histones  components o f c h r o m a t i n ,  genes  methionine  discussion).  structural  and  with  although  transported  Once bound t o DNA,  of histone  I t i s thought that  is initiated  rather than N - a c e t y l - s e r i n e still  isolation  Following  nucleus  where  a p p e a r t o be  judging  from the  i n pulse-chase  their they  stable  similar  experiments  (55,56). II.  Histone The  nature  although vealed that  the  by  the  while  Interactions with of  DNA  histone-DNA  asymmetric  and  basic regions  charge d i s t r i b u t i o n  of histones  could  A v a r i e t y of p h y s i c a l techniques  persion  (ORD)  histone  fragments  increased can  be  dichroism  ordering  observed with  increasing salt  approach to s t r u c t u r e i s to perform (60)  on  the  probability  along  with  (62),  Sung and  optical  isolated  structure  of h e l i x  model b u i l d i n g . Dixon  (61)  So, and  proteins.  magnetic rotatory  histones  resdis-  and  1-4,59).  (more a - h e l i c a l  concentrations.  An regions)  Another  theoretical calculations  formation  i n the  f o r example, L o u i e  Shih  DNA,  bind other  - nuclear  re-  (15,17,23)  interact with  i n s o l u t i o n ( f o r r e v i e w s see of histone  unclear,  of histones,  could  (CD),  - have b e e n a p p l i e d t o  still  suggestion  n e u t r a l , hydrophobic regions  (NMR), c i r c u l a r  Histones  interactions i s  sequence s t u d i e s , l e d t o the  the  onance  Other  and  Bonner  (63)  histones, et a l . have  built  -8-  a - h e l i c e s f o r the NH2-terminal r e g i o n s o f H2A,  H2B,  H3  and  H4, which f i t i n the major groove w i t h l y s y l and a r g i n y l c h a i n s i n a p p r o p r i a t e p o s i t i o n s t o i n t e r a c t w i t h DNA  side  phos-  phates. Histone-histone interactions  (long c o n s i d e r e d an  artifact  o f i s o l a t i o n procedures) can be observed when " g e n t l e " l a t i o n procedures  ( i . e . not a c i d e x t r a c t i o n ) are used.  i t has been r e p o r t e d t h a t H2A (33,34), w h i l e H3 and H4 may mers (32-34). NMR  iso-  and H2B  can i n t e r a c t as dimers  a s s o c i a t e as  Bradbury, Crane-Robinson  evidence t h a t the H3, H4  Thus  (H3)2 (H4) 2 t e t r a -  and co-workers  have  i n t e r a c t i o n i n s o l u t i o n occurs  a t the hydrophobic, c a r b o x y l - t e r m i n a l ends o f the m o l e c u l e s (64) . How  do the h i s t o n e s i n t e r a c t w i t h DNA?  Early  experiments  (65) on s a l t - t r e a t e d chromatin showed t h a t the h i s t o n e s e l u t e d i n an o r d e r HI, then H2A,  H2B,  then H3,  H4.  T h i s suggests  an o r d e r i n g f o r the " t i g h t n e s s " o f i n t e r a c t i o n w i t h DNA H3, H4  > H2A,  H2B  > Hi.  of  Indeed, removal o f H i r e s u l t s i n  n e g l i g i b l e change i n n u c l e o h i s t o n e c o n f o r m a t i o n as c h a r a c t e r i z e d by X-ray d i f f r a c t i o n , CD and ORD w h i l e H2A,  H2B,  data  (66-68).  So,  H3 and H4 are n e c e s s a r y t o m a i n t a i n b a s i c  chromatin s t r u c t u r e , H i may  be n e c e s s a r y as a  molecule f o r chromosome c o i l i n g  "crosslinking"  (69-71) and/or c o n d e n s a t i o n .  Very r e c e n t experiments, u t i l i z i n g n u c l e a s e d i g e s t i o n o f chromatin i n i n t a c t n u c l e i , i n d i c a t e t h a t t h e r e i s an o r d e r e d , r e p e t i t i v e a s s o c i a t i o n o f h i s t o n e s w i t h DNA.  These  results  -9-  p i c t u r e chromatin r e g i o n s o f DNA, molecules. The DNA  c o n s i s t i n g l a r g e l y o f 200  associated with  (probably)  base p a i r  long  eight histone  (See P a r t B of t h i s t h e s i s . )  c l o s e a s s o c i a t i o n and  s t o i c h i o m e t r y of h i s t o n e s  l e d t o the i d e a t h a t h i s t o n e s might be s p e c i f i c  s o r s c o n t r o l l i n g gene e x p r e s s i o n  (72).  I t i s now  and  repres-  thought  t h a t t h e r e are too few d i s t i n c t h i s t o n e types t o account  for  the v a r i a t i o n i n gene e x p r e s s i o n seen i n h i g h e r organisms; h i s t o n e s may  i n s t e a d be  s t r u c t u r a l components o f  chromatin  and/or "coarse" o r n o n - s p e c i f i c r e p r e s s o r s o f gene e x p r e s s i o n (104).  The  covalent modifications —  t i o n , methylation  etc. —  acetylation,  of the h i s t o n e s may  phosphoryla-  serve t o modu-  l a t e histone-DNA or h i s t o n e - h i s t o n e i n t e r a c t i o n s , and a l l o w changes i n the s t r u c t u r e or f u n c t i o n o f III.  C o v a l e n t M o d i f i c a t i o n s o f the  hence  chromatin.  Histones  Histone A c e t y l a t i o n As noted  earlier  l y s y l r e s i d u e s occur h i s t o n e s H2A, synthetic  H2B,  i n the i n t r o d u c t i o n , e - N - a c e t y l a t e d i n the b a s i c , NH2-terminal r e g i o n s o f  H3 and H4.  (73), enzymatic  enzyme A as a c e t a t e donor. been demonstrated  This a c e t y l a t i o n i s a post-  (74-76) p r o c e s s , w i t h a c e t y l  co-  Turnover o f e - N - a c e t y l groups  has  (77,78) and h i s t o n e d e a c e t y l a s e s have been  r e p o r t e d from c a l f thymus  (79,80).  Assuming the b a s i c r e g i o n s of h i s t o n e s i n t e r a c t w i t h a c e t y l a t i o n o f l y s y l r e s i d u e s i n these r e g i o n s e l i m i n a t e s  DNA,  -10-  positive  charges.  modulate the tivation can  or  alter  Adler  This could a l t e r  binding of histones chromatin  e t a l . (81)  effective circular  than  who  H4  t o DNA,  assembly.  i t s i n t e r a c t i o n with  histone  conformation  either  f o r gene  That a c e t y l a t i o n of a DNA  was  ac-  histone  recently reported  showed t h a t m o n o - a c e t y l a t e d H4  i n c h a n g i n g DNA  and  conformation,  by  is  less  monitored  by  dichroism.  T h e r e a r e many r e p o r t s w h i c h c o r r e l a t e h i s t o n e a c e t y l a tion  and  treated  i n c r e a s e d gene a c t i v a t i o n : lymphocytes  various c e l l direct  types  too  few  treated with  histone  o f gene e x p r e s s i o n the  (82), r e g e n e r a t i n g  specific  gene  Dixon  thesis,  (84)  and  However  no  since there f o r the  are  complexity "loosen"  a v a i l a b l e to other molecules  other  required to  possible functions of  obtained  "loosen"  DNA and  and  (78).  and  primary  evidence  synthesis, i n  that after  (to the unmodified  histone to  acetylation also  spermatocytes  a c e t y l a t i o n (to the  histone  histones prior  Histone  histone  cells,  the  (78) .  h i s t o n e H4  a rapid, obligatory diacetylation  a slower  deacetylation  In s p e r m a t i d  protamine  stem c e l l s  t h e r e was  f o l l o w e d by  DNA  two  associated with  large d i p l o i d and  be  r e p l a c e m e n t by  occurs,  i s evident;  and  o r g a n i s m s , a c e t y l a t i o n may  have been p r o p o s e d .  a c e t y l a t i o n may their  (83).  (77) ,  activation.  In t r o u t t e s t i s , acetylation  rat liver  species to account  i n higher  h i s t o n e s making the  phytohemagglutinin-  hormones  cause-effect relationship  probably  in  of  Louie  synH4,  t e t r a a c e t y l a t e d form)  form).  They  hypothesized  -11-  that  s u c h a c e t y l a t i o n , by n e u t r a l i z i n g  H4, a l l o w e d Histone The also  the c o r r e c t conformation  positive  f o r b i n d i n g t o DNA.  Phosphorylation  O-phosphorylation  of s e r y l  a p o s t s y n t h e t i c , enzymatic  phosphate donor.  residues  (85) p r o c e s s  Several histone  kinases,  d e n t o r i n d e p e n d e n t , have been i s o l a t e d plasm of  (1-4).  these  enzymes i s u n c l e a r ,  since isolated  for  many p r o t e i n k i n a s e s . lysyl  histones  phosphorylation  cells  Hi  histones.  and  are often  depen-  or cytoo f some  cytoplasmic  (H4) r e s i d u e s b y a p h o s p h o k i n a s e has been r e p o r t e d with  may s e r v e  different  (37). This phosphate  (86  histone  turnover  ).  functions f o r the  For histone H i , there  a p p e a r t o be two  a phosphorylation  o f almost a l l  from S phase through t o m i t o s i s which c o r r e l a t e s  chromosome c o n d e n s a t i o n ( i i ) lower l e v e l s  and m i t o s i s  (a few p e r c e n t )  the response o f c e l l s  gene  cAMP  More r e c e n t l y , t h e p h o s p h o r y l a t i o n  of phosphorylation:(i)  molecules  with  in  s i n c e they  i s a dynamic p r o c e s s ,  Phosphorylation  kinds  either  from n u c l e i  v a r i o u s histone phosphatases reported  different  u s i n g ATP a s  are often non-specific substrates  (HI) and h i s t i d y l  from carcinosarcoma  and  i n histones i s  However, t h e p h y s i o l o g i c a l s i g n i f i c a n c e  and  of  charges i n  t o hormones  (62,70,71,87-89)  of Hi phosphorylation (90,91) a n d r e s u l t a n t  activation. The  kinetics  of histone  H2A p h o s p h o r y l a t i o n  a rapid, obligatory phosphorylation correct  b i n d i n g o f H2A t o DNA  (35).  indicate that  may be n e c e s s a r y  f o r the  A s i m i l a r model has been  -12-  proposed f o r the b i n d i n g of protamines t o DNA by L o u i e & Dixon  (92). No f u n c t i o n i s e v i d e n t f o r the p h o s p h o r y l a t i o n  of h i s t o n e s H2B, H3 and H4.  I t was o r i g i n a l l y thought t h a t  h i s t o n e p h o s p h o r y l a t i o n was i n v o l v e d i n the " l o o s e n i n g " o f h i s t o n e s from DNA d u r i n g t h e i r replacement by protamines. However, h i s t o n e p h o s p h o r y l a t i o n does not occur t o a s i g n i f i c a n t e x t e n t i n spermatids process  (93,94), where t h i s replacement  occurs.  Poly-ADP-ribosylation Poly  (ADP-ribose) i s a polymer s y n t h e s i z e d from NAD by a  n u c l e a r enzyme which j o i n s ADP-ribose u n i t s i n a l " - 2 ' r i b o s e ribose linkage  ( f o r reviews,  see 95,96).  The s t r u c t u r e can  be r e p r e s e n t e d a s : n[Adenine-ribose-P-P-ribose-nicotinamide],  n £ 30  ADP-ribose Adenine  Adenine  I  Ribose  I  P-  T  I  Ribose  I  P—Ribose  P  GH  PD  (97) c l e a v e s a t GH.  Ribose P  GH  PD  P—Ribose  PD  c l e a v e s a t PD and a glycohydro-  T h i s polymer i s c o v a l e n t l y bonded  to the h i s t o n e s i n an a l k a l i - l a b i l e serine-phosphate  I  -P—Ribose  where venom phosphodiesterase lase  Adenine  I  i n h i s t o n e H i (98) .  linkage, possibly v i a a Further  localization  -13-  of p o l y  (ADP-ribose) may proceed f o l l o w i n g the p r e p a r a t i o n  of s p e c i f i c a n t i b o d i e s a g a i n s t t h i s polymer (99). The  f u n c t i o n o f t h i s polymer i n the nucleus i s unknown;  i t may r e g u l a t e DNA s y n t h e s i s , chromatin s t r u c t u r e o r couple NAD and energy metabolism t o DNA b i o s y n t h e s i s Protein  (95,96).  Methylation  L i k e a c e t y l a t i o n and p h o s p h o r y l a t i o n ,  the m e t h y l a t i o n  o f p r o t e i n s i s a p o s t s y n t h e t i c , enzymatic p r o c e s s S-adenosyl-L-methionine as methyl donor t i o n occurs  (100).  utilizing  T h i s methyla-  on c e r t a i n amino a c i d s i n the p r o t e i n backbone  —  x  N - m e t h y l - h i s t i d m e ; e-N-mono-, d i - and t r i m e t h y l - l y s i n e ; Q N -mono- and two d i m e t h y l - a r g i n i n e s , amino a c i d s glutamic  (100).  and a few o t h e r  minor  The c a r b o x y l groups o f a s p a r t i c and  a c i d can a l s o be found methylated as u n s t a b l e  methyl  esters. Three d i f f e r e n t c l a s s e s o f p r o t e i n methylases have been reported  (100):- p r o t e i n methylase I , a c y t o s o l enzyme which  methylates a r g i n i n e ; methylase I I , a p u r i f i e d c y t o s o l enzyme which methylates g l u t a m i c  and a s p a r t i c c a r b o x y l s ;  I I I , a n u c l e a r enzyme which methylates l y s i n e .  and methylase  Solubilization  of t h e l a s t enzyme from c h i c k n u c l e i has r e c e n t l y been r e ported  (101).  So f a r o n l y c e r t a i n p r o t e i n s o f s p e c i a l f u n c t i o n have been found methylated; the f u n c t i o n o f t h i s m e t h y l a t i o n i s unknown.  Methylation  o f the b a s i c  b a s i c i t y and h y d r o p h o b i c i t y  amino a c i d s i n c r e a s e s the  o f the p r o t e i n  (100);  modification  -14-  of  carboxyls  earlier  leads  thought  degradation protease that sin  1  to loss  (100) ; h o w e v e r ,  cytochrome  c  membrane i n t e r a c t i o n s .  in  proteins  the c o n t r a c t i l e  developmental been  (100)  (102).  lysyl  protein  (104),  p r o t e i n h y d r o p h o b i c i t y f o r subsequent Actin,  m y o s i n a n d some  bacterial  (100)  process  though f u n c t i o n a l and  i s unknown,  differences  identified  residues  — their  role  i n myosin m e t h y l a t i o n have  sites  i n histones  lysyl  absence  residues,  of e-N-methylation of  enzyme.  that  two e n z y m e s ,  Pea t i s s u e  Patterson  that other  and D a v i e s have  histones  (100)  lack  recognizing  indeed i s o l a t e d  are found i n  T  — N - m e t h y l - a r g i n i n e s and N - m e t h y l - h i s t i d i n e —  but the l o c a l i z a t i o n , g e n e r a l i t y , unclear.  This  H3 b u t n o t H4 (106) .  methyl-amino acids  G  note-  w o u l d l a c k t h e H4  a pea h i s t o n e methylase which m e t h y l a t e s There are r e p o r t s  is  being the  o f m e t h y l - l y s i n e i n p e a H4 ( 1 5 ) .  H3 o r H4 m i g h t be p r e s e n t .  It  spec-  i n the extent of methyla-  t h e most d r a m a t i c  p e a H4 m e t h y l a t i o n s u g g e s t s  recognizing  DeLange, Smith and c o w o r k e r s ,  H3 a n d H4 ( 1 2 - 2 1 ) .  there are differences  at these  complete  are  indicate  are also methylated  (105)  have  worthy t h a t  of  on t h e  reported.  and o t h e r s ,  tion  from  In the case o f rhodop-  and m y e l i n b a s i c  In the case o f the h i s t o n e s ,  ific  proteins  of chemically methylated proteins  m e t h y l a t i o n may i n c r e a s e  flagellar  I t was  more r e c e n t e x p e r i m e n t s  i s probably not true  (103) ,  charge.  that methylation might p r o t e c t  resistance  this  of a negative  Similarly,  reports  and s i g n i f i c a n c e o f that histones  these  H2A a n d H2B  -15-  are methylated  (100,107) c o u l d a r i s e from c o n t a m i n a t i o n o f  these h i s t o n e s (incomplete h i s t o n e f r a c t i o n a t i o n There a r e a c c o u n t s o f process  etc.).  h i s t o n e m e t h y l a t i o n as a s t a b l e  (108), w h i l e o t h e r s show i t t o be a dynamic e v e n t ,  w i t h m e t h y l groups t u r n i n g o v e r d u r i n g t h e c e l l c y c l e  (107,  109). H i s t o n e m e t h y l a t i o n appears t o be a l a t e e v e n t i n t h e H e l a c e l l c y c l e , o c c u r r i n g i n l a t e S and G synthesis  (100).  2  and h i s t o n e  Nuclear methylase I I I l e v e l s a l s o r i s e c o r -  respondingly i n G  2  (110).  I t i s p o s s i b l e then t h a t h i s t o n e  m e t h y l a t i o n has some r o l e i n subsequent or  a f t e r DNA  chromatin  condensation  mitosis. The system i n w h i c h we chose t o examine t h e p r o c e s s o f h i s t o n e  m e t h y l a t i o n was IV.  spermatogenesis  i n the d e v e l o p i n g t r o u t  testis.  Spermatogenesis In t h e immature t e s t i s , s p e r m a t o g o n i a l stem c e l l s -go  t h r o u g h a s e r i e s o f m i t o s e s t o produce p r i m a r y  spermatocytes.  These c e l l s , upon two f u r t h e r m e i o t i c d i v i s i o n s , g i v e r i s e t o h a p l o i d s p e r m a t i d s , which t h e n d i f f e r e n t i a t e f u r t h e r g e n e s i s ) t o g i v e mature sperm.  (spermio-  Whereas s p e r m a t o g e n e s i s  in  mammals i s a c o n t i n u o u s p r o c e s s w i t h a l l c e l l t y p e s p r e s e n t i n t h e t e s t i s a t a g i v e n time  ( 1 1 2 ) , t h e same p r o c e s s i n  Salmonids o c c u r s once y e a r l y w i t h t h e r e l a t i v e l y  synchronous  appearance o f c e l l t y p e s and i n c r e a s e i n t e s t i s w e i g h t As i s t h e c a s e f o r v e r t e b r a t e s (112), s p e r m a t o g e n e s i s Salmonid  (111). in  f i s h i s p r o b a b l y under the c o n t r o l o f p i t u i t a r y  -16-  gonadotrophins  (113).  Hence, the p r o c e s s can be induced i n  s e x u a l l y immature t r o u t salmon p i t u i t a r y e x t r a c t  (Salmo g a l r drier i i )  by i n j e c t i o n s w i t h  (114,115); when so induced, the  p r o c e s s i s more r a p i d and synchronous  than normal.  F o l l o w i n g the e a r l y stages o f spermatogenesis  (up t o  primary spermatocyte), spermiogenesis i s accompanied i n c e l l morphology — propulsive t a i l , c e l l volume. of  by changes  chromatin c o n d e n s a t i o n , development o f  l o s s o f cytoplasm and r e s u l t a n t decrease i n  These changes i n c e l l volume a l l o w s e p a r a t i o n  different cell  types on bovine serum albumin g r a d i e n t s  (116), a t e c h n i q u e used by Lam e t a l . (117) t o c h a r a c t e r i z e the  stages o f mouse spermatogenesis.  Louie and Dixon  used t h i s t e c h n i q u e t o l o c a l i z e , t o s p e c i f i c c e l l  (93,94)  t y p e s , the  b i o c h e m i c a l changes o c c u r r i n g d u r i n g spermatogenesis.  Thus,  most o f DNA, RNA and h i s t o n e s y n t h e s i s o c c u r s i n the l a r g e d i p l o i d stem c e l l s and primary spermatocytes.  These p r o c e s s e s  have stopped by the spermatid s t a g e , accompanied in  cell  RNA c o n t e n t and subsequent  by protamines. go  by a decrease  replacement o f h i s t o n e s  During t h i s replacement the protamines  under-  an o b l i g a t o r y p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n (92),  n e c e s s a r y perhaps f o r b i n d i n g t o DNA and subsequent t i o n o f chromatin.  As noted e a r l i e r i n t h i s  condensa-  introduction,  h i s t o n e p h o s p h o r y l a t i o n and a c e t y l a t i o n a r e a s s o c i a t e d mainly w i t h the l a r g e d i p l o i d stem c e l l s and spermatocytes  (78,93,94),  w h i l e i n spermatids no p h o s p h o r y l a t i o n , b u t some a c e t y l a t i o n o c c u r s , perhaps as a n e c e s s a r y p r e l u d e t o the replacement o f  -17-  histones  by  Since histone in  protamine. the  sites,  a c e t y l a t i o n and  trout testis,  we  and  the  precursors  —  localizations  are w e l l  i n spermatogenesis.  ready  ability  cellular  phosphorylation  t i s s u e a v a i l a b l e , the  s y n c h r o n y and  and  easy p r e p a r a t i o n  separation  of c e l l  a l l are  The  of  the  of  role  of^  large quantities chromatin,  different  suspensions to  of  characterized  were i n t e r e s t e d i n l o o k i n g a t t h e  histone methylation of  kinetics  cell  the  types,  incorporate radioactive  f u r t h e r good r e a s o n s  for using  this  system. Part A of kinetics The H3  H4  indicate that  are methylated,  determined  for other  associated with ploid  cells.  s p e r m a t i d s and There testis H4  t h e s i s r e p o r t s on  of histone methylation  results and  this  DNA  is negligible cells.  The  histone  H4  synthesis.  some  trout  residues  testis.  of  This methylation synthesis  i s probably  not  replacement of histones turnover kinetics  indicate that methylation  after  lysyl  and  histones  i n p o s i t i o n s homologous t o  This process the  sites  i n developing  specific  organisms. and  the  of histone of methyl i s a late  i n the  those  occurs large d i -  associated by  protamines.  methyl groups  labelling event  with  of  i n the  in trout  histone cell  cycle  -18-  MATERIALS AND METHODS I.  Chemicals (a)  and A b b r e v i a t i o n s  Chemicals  A l l chemicals o b t a i n e d commercially were of the h i g h e s t p u r i t y o r reagent grade. Instruments  Sequencer Grade chemicals  (Beckman  I n c . o r P i e r c e Chemical Co.) were used f o r p r o -  t e i n sequence s t u d i e s w i t h the Beckman 890 Sequencer. R a d i o a c t i v e compounds: - L - [ m e t h y l - C ] m e t h i o n i n e 14  mCi/mmole), L-[methyl- H]methionine 3  lysine  (5 Ci/mmole), L - [ ^ C ]  (340 mCi/mmole), D L - [ 4 . 5 - H ] l y s i n e 3  L-[5- H]arginine 3  S e a r l e Corp.  (56  1  (20 Ci/mmole) and  (8.9 Ci/mmole) - were o b t a i n e d from Amersham-  S t a r c h f o r g e l e l e c t r o p h o r e s i s was o b t a i n e d  from Connaught L a b o r a t o r i e s and E l e c t r o s t a r c h Company. (b)  Abbreviations  dansyl chloride:  l,dimethylaminonaphthalene-5-sulfonyl  chloride POPOP: PPO:  1,4-Bis-(5-phenyloxazol-2-yl)-benzene 2,5-diphenyloxazole  TCA-tungstate:  5% t r i c h l o r o a c e t i c a c i d , 0.25% sodium  t u n g s t a t e , pH 2 TMKS b u f f e r : KC1  Tris-HCl  (25 mM) and sucrose  (50 mM, pH 7.4), MgClz (1 mM),  (0.25 M)  monomethyl-lysine: N -methyl-histidme:  e-N-monomethyl-L-lysine 3-methyl-histxdine  -19-  dimethyl-lysine: trimethyl-lysine: II.  e-N-dimethyl-L-lysine £-N-trimethyl-L-lysine  I s o l a t i o n and C h a r a c t e r i z a t i o n o f iri v i v o [ ''C]methyl1  ated Trout T e s t i s (a)  [ **C]methyl 1  Histones l a b e l l i n g of histones  T e s t e s were o b t a i n e d from rainbow t r o u t i n which sperma t o g e n e s i s had been induced by twice-weekly salmon p i t u i t a r y e x t r a c t s (114).  i n j e c t i o n of  T e s t e s were s c i s s o r - m i n c e d  i n 2 volumes o f TMKS b u f f e r c o n t a i n i n g 0.1% g l u c o s e , and were then g e n t l y homogenized i n a P o t t e r - E l v e h j e m homogenizer. The  resulting c e l l  suspension was f i l t e r e d through  cheese-  c l o t h , and was then i n c u b a t e d w i t h 20 yCi/ml o f L - [ m e t h y l ''C] methionine  1  a t 16-18°C f o r up t o 7 hours i n t h e presence  o f 100 U/ml o f p e n i c i l l i n and s t r e p t o m y c i n  (Baltimore B i o -  l o g i c a l ) , and supplemented w i t h one t e n t h volume o f T r i s b u f f e r e d Waymouth's medium (118) as d e s c r i b e d by L o u i e and Dixon  ( 84).  C e l l s were c o l l e c t e d by c e n t r i f u g a t i o n , homo-  g e n i z e d v i g o r o u s l y i n TMKS b u f f e r , and n u c l e i were p e l l e t e d a t 1,000 g f o r 10 min. A f t e r two more washings o f n u c l e i w i t h TMKS, chromatin was prepared by washing n u c l e i w i t h 0.15 M NaCl,  further  20 mM EDTA and then t h r e e times i n 10 mM  T r i s pH 8.0 ( 9 ) .  The h i s t o n e s were e x t r a c t e d from t h e  v i s c o u s chromatin p e l l e t w i t h 0.4 N H S O i t 2  w i t h 4 volumes o f e t h a n o l a t -20°C.  and p r e c i p i t a t e d  The h i s t o n e s were then  -20-  d i s s o l v e d i n w a t e r , l o a d e d on a c a r b o x y m e t h y l - c e l l u l o s e column (Bio-Rad L a b o r a t o r i e s ) and h i s t o n e s HI and H6 were p u r i f i e d by L i C l g r a d i e n t e l u t i o n  (9  ).  A f t e r the r e m o v a l o f  his-  tones H6 and H i , t h e o t h e r h i s t o n e s were e l u t e d from t h e column w i t h 0.2  N H C l , c o n c e n t r a t e d by l y o p h i l i z a t i o n ,  s e p a r a t e d on l o n g B i o - G e l P-10 i n 0.01  N HCl  ( 40).  (Bio-Rad L a b o r a t o r i e s ) columns  When r e q u i r e d , p r o t a m i n e was  from t h e h i s t o n e s on a s h o r t e r (2 x 100 cm) p r i o r to t h i s step.  o f h i s t o n e were c o u n t e d i n B r a y ' s s o l u t i o n  (b)  P-10  For r a d i o a c t i v i t y a n a l y s i s ,  t e i n c o n c e n t r a t i o n was  monitored  and S m i t h i e s  aliquots  (119), w h i l e p r o -  monitored  nm.  by e l e c t r o p h o r e s i s on  (120).  urea-  as d e s c r i b e d by Sung  H i s t o n e samples were e l e c t r o p h o r e s e d  a t 6 v o l t s / c m , 30 m i l l i a m p e r e s w i t h w a t e r - c o o l e d  gel trays  G e l s were h o r i z o n t a l l y t r i s e c t e d , and m i d d l e  bottom s l i c e s were s t a i n e d 40 min w i t h 0.1% t a i n i n g C o C l , and d e s t a i n e d i n N 2  Determination  H2SO4  of methylated  In p r e l i m i n a r y analyses, was  column,  by absorbance a t 230  aluminum l a c t a t e s t a r c h g e l s , p r e p a r e d  (c)  separated  Starch gel electrophoresis  H i s t o n e p u r i t y was  a t 4°C.  and  or  Amido B l a c k con-  (120).  amino a c i d s  [ ''C]methyl-labelled p r o t e i n 1  h y d r o l y z e d i n 6 N HCl i n v a c u o , a t 110°C  f o r 22  hours.  H y d r o l y s a t e s were e l e c t r o p h o r e s e d on Whatman 3 MM paper a t  -21-  pH  6.5  (pyridine/acetic  a c i d / w a t e r , 100:4 :900) w i t h  methio-  n i n e and v a r i o u s m e t h y l - l y s i n e s t a n d a r d s as f o l l o w s : methyl-lysine  (Sigma), d i m e t h y l - l y s i n e  trimethyl-lysine Labelled  (a g e n e r o u s  gift  mono-  (Cyclo Chemical), and  from Dr. Robert  DeLange).  amino a c i d s were l o c a t e d b y a u t o r a d i o g r a p h y o n  Kodak B l u e B r a n d  X-ray f i l m  a n d e l u t e d w i t h 25% a c e t i c  R a d i o a c t i v i t y was d e t e r m i n e d o n a l i q u o t s Later,  acid  a n a l y z e r w i t h Durrum DC1A r e s i n  a t 41°C i n 0.43 M c i t r a t e ,  mine r a d i o a c t i v i t y  i n the methyl  e f f l u e n t was d i v e r t e d  (d)  dissolved  pH 6.1.  amino a c i d s ,  from t h e r e a c t i o n c o i l  f r a c t i o n s were c o l l e c t e d . an a l i q u o t  solution.  h y d r o l y s a t e s were c h r o m a t o g r a p h e d o n a  T e c h n i c o n amino a c i d Instruments)  i n Bray's  acid.  (Durrum  To d e t e r -  t h e column a n d 1.0 m l  R a d i o a c t i v i t y was d e t e r m i n e d o n  i n Aquasol  (New E n g l a n d  Determination o f c e l l u l a r  Nuclear).  L-methionine  and S-  adenosyl-L-methionine Testis  cells  were i n c u b a t e d i n TMKS - 0.1% g l u c o s e w i t h  L-[methyl- ^C]methionine 1  f o r 10-30 m i n , t h e n i n t r a c e l l u l a r .  [ "*C] m e t h i o n i n e a n d [ **C] S - a d e n o s y l - L - m e t h i o n i n e 1  1  m i n e d b y t h e method o f S a l v a t o r e e t a l . (121). cedure, p e r c h l o r i c  acid  applied  (X8) ( B i o - R a d )  t o Dowex-1  2 N H SOi» 2  extracts of collected  ( f o r methionine)  L-methionine) .  were In t h i s  cells  columns and e l u t e d  and 6 N  H2SO4  deterpro-  were with  ( f o r S-adenosyl-  -22-  III.  S i t e s of i n vivo Methylation i n Trout T e s t i s Histones (a)  Automated p r o t e i n  Samples of i n 0.6  ml  sequencing  [^C]methyl l a b e l l e d histone,  of N a c e t i c a c i d , were s u b j e c t e d  t i o n s i n a Beckman 890 reagents as d e s c r i b e d  protein  0.5-1.0 ymoles  to Edman degrada-  sequencer, u s i n g  by N i a l l e t a l . (122).  volatile  In t h i s DMAA  program, p h e n y l i s o t h i o c y a n a t e b u f f e r e d  with  dimethylallylamine  (DMAA) t r i f l u o r o a c e t i c a c i d i s r e a c t e d  with p r o t e i n ,  followed  by a benzene wash, double c l e a v a g e w i t h  heptafluorobutyric  a c i d , and  d e r i v a t i v e with  e x t r a c t i o n o f the t h i a z o l i n o n e  butyl chloride. of the  The  2-anilino-5-thiazolinone  amino a c i d s were then c o n v e r t e d to the  thiohydantoin  (PTH)  80°C f o r 10 min  d e r i v a t i v e s by  under N2  (123).  i d e n t i f i e d on a Beckman 120C a f t e r h y d r o l y s i s o f the PTH f o r 24 hr o f the  (124).  3-phenyl-2-  incubation  i n N HCl  at  Amino a c i d s were then  o r Technicon amino a c i d d e r i v a t i v e s i n 6 N HCl  R a d i o a c t i v i t y was  2-anilino-5-thiazolinone  redissolved Dixon  derivatives  determined on  a t 140°C aliquots  d e r i v a t i v e s d r i e d down  i n Bray's s o l u t i o n , as d e s c r i b e d  analyser  and  by Candido  and  ( 22) „  (b) 50 mg  [ C]methyl 1U  peptides of histone  of histone  H3  (180,000 cpm)  a l i q u o t s o f N-bromosuccinimide  was  H3 reacted  (6 mg/ml i n 50%  over 2 hours at 20°C u n t i l t h e r e was  no  w i t h 1 ml  a c e t i c acid)  further increase  in  -23-  A2 6 0 .  After  a r a t e d on in  25%  lyophilization,  a Sephadex G-50  acetic  acid.  lyophilization, umn.  The  the  resulting  column  p e p t i d e s were  (Pharmacia),  monitored  by  single radioactive peptide  composition  corresponding  a c i d s o f h i s t o n e H3 with  30 mg  of maleic  essentially lated in  anhydride  then  p y r i d i n e - a c e t a t e pH  x 200  cm,  t i o n s were p o o l e d , Whatman 3 MM  by  40  amino  (c) 70 150  mg  i n 10%  and  M NaHCG-3, pH  trypsin  8.5,  a Sephadex G-10  and  acid.  The  (1:50  demaleylated  acetic  The  maley-  by  weight)  i n 0.05 The  M  resulting (Pharmacia)  Radioactive  chromatographed  frac-  on  with  eluted with  radioactive peptides, located  Kodak B l u e B r a n d X - r a y 25%  acetic  Tryptic peptides of mg  maleylated  h o u r s a t 37°C.  concentrated  ( 12,126).  and  next  paper i n p y r i d i n e : b u t a n o l : a c e t i c acidswater,  autoradiography  cut out  nm.  t o the N-terminal  i n 0.1  f o r 40  and  amino  p e p t i d e s were c h r o m a t o g r a p h e d on c o l u m n , 2.5  col-  of  digested with  3.2  by  so i s o l a t e d ,  was  M ammonium b i c a r b o n a t e  15:10:3:12  same  a s d e s c r i b e d by B u t l e r e t a l . (125).  p e p t i d e was  0.1  (17-21),  the  reaction with ninhydrin  measurement o f s u b s e q u e n t a b s o r b a n c e a t 570  acid  cm,  r a d i o a c t i v e peak, c o n c e n t r a t e d  w a s - t h e n r e c h r o m a t o g r a p h e d on  P r o t e i n was  The  2 x 180  sep-  o f h i s t o n e H4  of maleic  [  l l f  were  acid.  C]methyl l a b e l l e d  (150,000 cpm)  anhydride  film,  was  histone  maleylated  (125), d i g e s t e d w i t h  with  trypsin  H4  -24-  (1:50) a n d d e m a l e y l a t e d cribed  i n pyridine-acetate  above f o r h i s t o n e  H3.  pH 3.2 a s d e s -  The r e s u l t i n g p e p t i d e s  were  c h r o m a t o g r a p h e d o n a S e p h a d e x G-25 c o l u m n  i n 25% a c e t i c a c i d ,  and  and c h r o m a t o g r a p h e d  on  t h e r a d i o a c t i v e peak was c o n c e n t r a t e d Whatman  3 MM p a p e r a s d e s c r i b e d  The  single radioactive peptide  25%  acetic acid.  (d) tryptic  Sequence  above f o r h i s t o n e  H3.  was c u t o u t and e l u t e d  determinations  on  [^C]methyl  with  labelled  peptides  A m o d i f i c a t i o n o f t h e Edman d e g r a d a t i o n f o r m e d on 0.1 t o 0.2 y m o l e s o f p e p t i d e , Candido and Dixon coupling  with  ( 40 ) .  Briefly,  phenylisothiocyanate  (127) was  as d e s c r i b e d  per-  by  the procedure  involves  i n pyridine +  N-ethyl-  morpholine b u f f e r , drying  u n d e r vacuum, c y c l i z a t i o n i n  anhydrous t r i f l u o r o a c e t i c  acid, drying,  and e t h y l  acetate  e x t r a c t i o n o f t h e t h i a z o l i n o n e d e r i v a t i v e o f t h e amino a c i d . Before  e a c h Edman s t e p ,  an a l i q u o t o f p e p t i d e  f o r amino a c i d a n a l y s i s dansylation (129).  according  aliquot in  (128) a s m o d i f i e d  A l i q u o t s of the released  redissolving The  ( s u b t r a c t i v e Edman s e q u e n c i n g ) o r  t o Gray  d e r i v a t i v e s were a n a l y z e d  by H a r t l e y  2-anilino-5-thiazolinone  forradioactivity  by d r y i n g and  i n Bray's s o l u t i o n .  dansylation  procedure  i n 0.1 M NaHC03 w i t h  an e q u a l  was r e m o v e d  involved  dansyl  reacting the peptide  chloride  (2.5 mg/ml)  v o l u m e o f a c e t o n e a t 37°C f o r 2 h o u r s ,  then  -25-  The d a n s y l d e r i v a t i v e o f the N H 2 - t e r m i n a l  d r y i n g i n vacuo. amino a c i d was for  16 hr) and was  graphy ing  Co.,  ( i n 6 N HC1  i d e n t i f i e d by two-dimensional  (129) on 5 x 5 cm polyamide  sheets  at  dimension  S o l v e n t systems  (Cheng C h i n T r a d -  used were as  - S o l v e n t 1, 1.5%  110°C  chromato-  T a i p e i ; d i s t r i b u t e d by G a l l a r d - S c h l e s i n g e r ,  P l a c e , N.Y.). 1st  l i b e r a t e d by h y d r o l y s i s  Carle  follows:  formic a c i d  2nd dimension - S o l v e n t 2, b e n z e n e : a c e t i c a c i d ,  9:1  S o l v e n t 3, e t h y l a c e t a t e : m e t h a n o l : a c e t i c a c i d , 20:1:1 IV.  H i s t o n e M e t h y l a t i o n i n D i f f e r e n t C e l l Types from ing  Develop-  Trout T e s t i s  (a)  C e l l separations  Testis c e l l induced t e s t i s  suspensions were p r e p a r e d from  hormonally-  (as i n M a t e r i a l s & Methods, s e c t i o n I I (a))  and a l i q u o t s o f 0.5 ml  (1 t o 7 x 1 0  8  c e l l s ) were then  incu-  bated w i t h 5 yCi/ml o f L - [ ^ C ] l y s i n e and 2 mCi/ml o f L1  [methyl- H]methionine 3  a t 16°C  f o r 2 hours.  l e c t e d by c e n t r i f u g a t i o n , resuspended saline  117).  i n phosphate-buffered  (0.9% NaCl) and a 15 ml a l i q u o t c o n t a i n i n g  c e l l s was 1-3%  C e l l s were c o l -  a l l o w e d t o sediment  a t u n i t g r a v i t y on a 600  bovine serum albumin g r a d i e n t f o r 255 min Then  7 to 9 x  a t 4°C  8 5 f r a c t i o n s o f 7 ml each were c o l l e c t e d  the g r a d i e n t i n 45 minutes,  10  ml, ( 93 /  from  so t h a t the t o t a l c e l l s e p a r a t i o n  7  -26-  time was Dixon  300 minutes  ( 93). (b)  (5 h o u r s ) , as d e t a i l e d by L o u i e  C e l l s were counted  and  on a hemocytometer.  Starch gel electrophoresis for histones  C e l l s o f d i f f e r e n t t y p e s , c o l l e c t e d from the g r a d i e n t , were f i l t e r e d onto g l a s s f i b e r f i l t e r s AP  20-025-00), washed w i t h phosphate-buffered  f i x e d with ethanol. in  ( M i l l i p o r e Corp.,  0.4  The  saline,  and  f i l t e r s were then e x t r a c t e d d i r e c t l y  N HCl i n the s l o t s o f a u r e a - l a c t a t e s t a r c h g e l and  e l e c t r o p h o r e s e d , as d e s c r i b e d by L o u i e and Dixon  (93).  Gels were h o r i z o n t a l l y t r i s e c t e d ; the bottom s l a b s t a i n e d w i t h cobalt-Amido (120), w h i l e the middle  Black  s l a b was  was  (Schwartz-Mann) f o r h i s t o n e s used f o r a n a l y s i s o f r a d i o -  a c t i v i t y i n the h i s t o n e s , as d e s c r i b e d below. (c) 0.5  Pulse-chase  turnover  ml of a t e s t i s c e l l  i n c u b a t e d w i t h 100  suspension  (5 x 1 0  yCi/ml o f D L - [ 4 , 5 - H ] l y s i n e  L-[methyl- ^C]methionine 1  above.  experiments  3  f o r 2 hours a t 16°C  8  cells)  and  50  was  uCi/ml  as d e s c r i b e d  C e l l s were then c o l l e c t e d by c e n t r i f u g a t i o n and r e -  suspended i n 8 ml o f TMKS - 0.1%  glucose with  streptomycin,  p e n i c i l l i n , T r i s - b u f f e r e d Way-mouth* s medium (see M a t e r i a l s and Methods, s e c t i o n I I ( a ) ) , and supplemented w i t h 1 L - l y s i n e and L-methionine present.  During  mM  to d i l u t e r a d i o a c t i v e precursors  f u r t h e r i n c u b a t i o n a t 16°C,  1 ml  aliquots  -27-  (5 x 1 0  7  c e l l s ) were f i l t e r e d onto g l a s s f i b e r  filters,  c e l l s were f i x e d and r a d i o a c t i v i t y i n t h e h i s t o n e s was d e t e r mined on s t a r c h g e l s as d e s c r i b e d below. C e l l v i a b i l i t y d u r i n g t h i s f u r t h e r "chase" i n c u b a t i o n was  m o n i t o r e d as f o l l o w s .  A f t e r the i n i t i a l  c e n t r i f u g a t i o n and r e s u s p e n s i o n ,  2 hr incubation,  a n a l i q u o t o f t h e c e l l s was  f u r t h e r i n c u b a t e d w i t h 50 y c i / m l o f L - [ H ] a r g i n i n e and t o t a l 3  r a d i o a c t i v i t y incorporated into c e l l n u c l e i at varying was  times  d e t e r m i n e d as d e s c r i b e d below. (d)  Radioactivity analysis  T o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o c e l l n u c l e i from c e l l s e p a r a t i o n g r a d i e n t f r a c t i o n s was d e t e r m i n e d by p r e c i p i t a t i o n w i t h T C A - t u n g s t a t e on g l a s s f i b e r f i l t e r s ( 9 3 ) . R a d i o a c t i v i t y i n t h e h i s t o n e s was d e t e r m i n e d from t h e m i d d l e s l a b o f t h e s t a r c h g e l , w h i c h was c u t i n t o 2 mm These s l i c e s were i n c u b a t e d  slices.  i n s c i n t i l l a t i o n v i a l s w i t h 0.5  ml NCS r e a g e n t (Amersham-Searle) o v e r n i g h t , t h e n 5 m l o f toluene s c i n t i l l a t i o n f l u i d  ( t o l u e n e + 0.01% POPOP and 0.4%  PPO) was added and t h e v i a l s i n c u b a t e d a t 45°C f o r 3 h o u r s p r i o r t o c o o l i n g and c o u n t i n g V.  K i n e t i c s o f Methyl (a)  ( 93 ) .  L a b e l l i n g o f Histone  H4  P r e p a r a t i o n o f [^C] m e t h y l l a b e l l e d h i s t o n e H4  A t e s t i s c e l l suspension  was i n c u b a t e d w i t h 20 y C i / m l  o f L - [ m e t h y l - ' ' C ] m e t h i o n i n e , supplemented w i t h 1  penicillin,  -28-  s t r e p t o m y c i n and  Waymouth's medium minus methionine  & Methods, s e c t i o n II  (a)] and  at varying  to 12 h r , a l i q u o t s were taken and  histone  by a c i d e x t r a c t i o n o f chromatin, and carboxymethylcellulose and  and  Bio-Gel  time i n t e r v a l s up H4 was  prepared  chromatography  P-10  columns  R a d i o a c t i v i t y i n the m o d i f i e d  species  on  [Materials  Methods, s e c t i o n II ( a ) ] . (b)  H i s t o n e H4  of histone  samples, prepared a f t e r v a r y i n g  i n c u b a t i o n , were e l e c t r o p h o r e s e d R a d i o a c t i v i t y i n the a c e t y l a t e d was  (Materials  then determined, as d e s c r i b e d  from s t a r c h g e l s l i c e s  times of  on u r e a - l a c t a t e and  starch gels.  p h o s p h o r y l a t e d H4  by L o u i e and  [ M a t e r i a l s and  H4  Dixon  species ( 84),  Methods, s e c t i o n IV  (d)].  -29-  RESULTS I.  I s o l a t i o n and Trout At  Testis  the  of  and  - from v a r i o u s  m e t h y l amino a c i d s  H4  -  derivatives  and  (100)  In d e v e l o p i n g  and  as  this  had  sperm-specific  We  H2A, of  is a  were  found.  synchronous  s p e r m a t o c y t e s -*• striking  morphological  l o s s of  histones In t h i s  a c e t y l a t i o n and  characterized  were t h e r e f o r e  r o l e of histone  also  -  methylation  by  model  phos-  Dixon  interested in i n the  and  and  the  development  of  tissue. This  first  section deals  histones  are  present,  following  1  .  (43)  protamines.  histone  been e x t e n s i v e l y  ( 28 , 62)  n a t u r e and  there  - spermatogonia  system f o r d i f f e r e n t i a t i o n , phorylation  histones  dimethyl-arginine  N -methyl-histidine  changes, i n c l u d i n g the  the  of  trimethyl-lysine  N - m e t h y l and  sperm - a c c o m p a n i e d by  appearance of  methylation  were  Furthermore, a wide v a r i e t y  trout testis,  cells  biochemical  coworkers  (100)  e-N-mono,di- and  well  spermatids  Methylated  started, there  ( 12 , 17 , 21 ,100) , b u t  sources.  ( 12 , 21 , 1 0 0 ) , as  development of  in vivo  i n v e s t i g a t i o n was  postsynthetic  p r e d o m i n e n t l y H3  of  Histones  time t h i s  many r e p o r t s  H2B  Characterization  methylated,  ''C] m e t h i o n i n e .  and  incubation  with  i d e n t i f y i n g which  w h i c h m e t h y l amino a c i d s of  testis  cells  with  testis are  L-[methyl-  /  -30-  (a)  Which h i s t o n e s a r e methylated  Histones  were prepared  from such an i n c u b a t i o n , as des-  c r i b e d i n M a t e r i a l s and Methods.  F i g u r e 1 shows the L i C l  g r a d i e n t e l u t i o n o f h i s t o n e s H6 and H i from a carboxymethylc e l l u l o s e column.  Amino a c i d a n a l y s e s  o f H6 and H i show  t h a t n e i t h e r h i s t o n e c o n t a i n s methionine, y e t H6 showed some [ ''C] i n c o r p o r a t i o n and so was p o s s i b l y m e t h y l a t e d . 1  When protamine was p r e s e n t  i n the sample, i t was sep-  a r a t e d from h i s t o n e s on a B i o - G e l P-10 column  ( F i g . 2 ) . The  [ *C] i n c o r p o r a t i o n i n t o protamine proved t o be as N - t e r m i n a l lf  methionine  (as judged by 99% l o s s o f r a d i o a c t i v i t y a f t e r 1  Edman d e g r a d a t i o n ) , The  as d e s c r i b e d by Wigle and Dixon  histones, recovered  (130) .  from the c a r b o x y m e t h y l c e l l u l o s e  column, were then f r a c t i o n a t e d on a l o n g B i o - G e l P-10 column (Fig. 3). Histones  H3 and H4 were t h e predominant l a b e l l e d  s p e c i e s , w i t h some perhaps i n H2B. t a i n s roughly  S i n c e h i s t o n e H2B con-  t h e same p r o p o r t i o n o f i n t e r n a l methionine as  H3 and H4, i t was s u r p r i s i n g t h a t so much more [^C]  label  appeared i n H3 and H4. (b) The  Which methylated b a s i c amino a c i d s a r e p r e s e n t nature  o f the [^C] l a b e l i n c o r p o r a t e d i n t o each o f  the h i s t o n e s was next examined, by a c i d h y d r o l y s i s o f the h i s t o n e s and p r e l i m i n a r y f r a c t i o n a t i o n o f amino a c i d s by pH 6.5 e l e c t r o p h o r e s i s .  The methyl b a s i c amino a c i d s a r e s t a b l e  -31-  F I G . 1. L i C l gradient separation of [ C]methyl l a b e l l e d h i s tones. T e s t i s c e l l s were i n c u b a t e d w i t h [ m e t h y l - "*C] m e t h i o n i n e and t h e h i s t o n e s were p r e p a r e d and f r a c t i o n a t e d on a 2 x 20 cm c a r b o x y m e t h y l - c e l l u l o s e c o l u m n w i t h a 400 m l 0.15 t o 0.75 M L i C l gradient. P r o t e i n was m o n i t o r e d by f o l l o w i n g t h e A 2 3 0 . F o l l o w i n g t h e e l u t i o n o f H6, t h e g r a d i e n t was s t o p p e d and t h e r e s t o f t h e h i s t o n e s were washed f r o m t h e c o l u m n w i t h 0.2 N HCl. 1 1 1  1  -32-  F I G . 2. The s e p a r a t i o n o f [ ^ C ] m e t h y l l a b e l l e d h i s t o n e s f r o m protamines. ' ' C - l a b e l l e d h i s t o n e s and p r o t a m i n e s were c h r o m a t o g r a p h e d on a 2 x 100 cm B i o - G e l P-10 c o l u m n i n 0.01 N H C l . P r o t e i n was m o n i t o r e d b y A 3 0• 1  2  -33-  c  .0 C  (TJ  r :  « w  O _ 2000  8  •5 H1200  120  FRACTION  NO.,  160  200  6ml.  FIG. 3. P r e p a r a t i o n o f [ ^ C ] m e t h y l l a b e l l e d h i s t o n e f r a c t i o n s on Bxo-Gel P-10. The h i s t o n e s (minus H I , H6), r e c o v e r e d from Bio-Gel P-10 chromatography ( F i g . 2 ) , were chromatographed on a l o n g (3 x 360 cm) B i o - G e l P-10 column i n 0.01 N HC1. Protein was m o n i t o r e d by A 3 . 2  0  o  fl) Q. 5 o  400  80  5  -34-  to  acid  hydrolysis  (100) , and move r a p i d l y  c a t h o d e , whereas m e t h i o n i n e near the o r i g i n  (Fig. 4).  i s less  highly  I t was e v i d e n t  h i s t o n e s H I , H2A a n d p r o t a m i n e  towards t h e charged  and s t a y s  ( F i g . 4) t h a t  c o n t a i n e d no m e t h y l a t e d  basic  amino a c i d s , w h e r e a s H6, H2B, H3 a n d H4 d i d . The  p r o p o r t i o n o f [ "*C]methyl 1  i n t e r n a l methionine  i s surprisingly  accounts  f o r the high incorporation  to  One e x p l a n a t i o n o f t h i s  H2B.  [methyl- ^C]methionine 1  donor  S-adenosyl  amino a c i d high  (90%) and relative  i s t h a t most o f t h e  i s converted t o the methyl  [methyl- ^C]methionine  (SAM).  1  Examination  of  the levels  et  a l . (121) show t h a t t h i s may i n d e e d be t h e c a s e , a s g r e a t e r  than  of methionine  1  i n t o H3 a n d H4  result  i n the c e l l s  t o [ ^C]  80% o f t h e c e l l This result  inhibit  protein  methionine cell  radioactivity  o b v i a t e d t h e need synthesis  incorporation.  viability  a n d SAM by t h e method o f S a l v a t o r e  i s r e c o v e r a b l e a s SAM. t o use cycloheximide t o  and t h u s m i n i m i z e  Such t r e a t m e n t would have  over the long incubation  experiments. . To i d e n t i f y  • which  internal  methyl  .  times used  [ '*C] 1  lowered  f o r some  •  amino a c i d s were p r e s e n t ,  acid  h y d r o l y s a t e s o f h i s t o n e s were c h r o m a t o g r a p h e d  on a T e c h n i c o n  amino a c i d  and Methods.  A  a n a l y z e r as d e s c r i b e d  i n Materials  s t a n d a r d r u n i s shown i n F i g . 5.  h i s t o n e were f o u n d t o c o n t a i n lysine,  b u t no r a d i o a c t i v i t y  [  llf  H y d r o l y s a t e s o f whole,  C ] mono-, d i - a n d t r i m e t h y l -  or ninhydrin positive  material  -35-  FIG. 4. The presence o f [ ''C]methionine and [ C ] m e t h y l amino a c i d s i n the h i s t o n e s . A c i d h y d r o l y s a t e s of the h i s t o n e s (6N HCl, 20 hr) were e l e c t r o p h o r e s e d a t pH 6.5 on Whatman 3 Mil paper as d e s c r i b e d i n the t e x t . C - r a d i o a c t i v i t y was l o c a t e d by autoradiography on Kodak Blue Brand X-ray f i l m . [ *C]methyl amino a c i d s moved r a p i d l y w i t h methyl green marker towards the cathode (spots at the top o f the chromatogram), whereas [ C ] methionine remained near the o r i g i n . 1  ll4  1 4  1!  I l +  -36-  ^  0.2  240  270  300  330  TIME, min  F I G . 5. S e p a r a t i o n o f e - N - m e t h y l - l y s i n e s (mono, d i - and t r i m e t h y l d e r i v a t i v e s ) on a T e c h n i c o n amino a c i d a n a l y s e r . Methylamino a c i d s t a n d a r d s were c h r o m a t o g r a p h e d on Durrum DC1A resin (Durrum I n s t r u m e n t s ) a t 41°C i n 0.43 M c i t r a t e , pH 6.1. A flow r a t e o f 12.5 m l / h r was u s e d f o r t h e 55 x 0.9 cm a n a l y s e r column. The e l u t i o n p o s i t i o n s o f h i s t i d i n e ( H i s ) and N - m e t h y l h i s t i d i n e [ H i s ( x M e ) ] a r e marked by a r r o w s . Ammonia, a r g i n i n e and i t s d e r i v a t i v e s e l u t e l a t e r f r o m t h e c o l u m n . T  -37-  was  found  tions  i n the N -methyl-histidine or methyl-arginine  o f t h e chromatogram. Histone  in  the r a t i o  H4 c o n t a i n e d dues. of  H3 c o n t a i n e d  mono-, d i - a n d t r i m e t h y l - l y s i n e  0.49:0.61:0.18  residues per molecule.  Despite  the finding,  after  i n h i s t o n e H6,  no n i n h y d r i n - p o s i t i v e m a t e r i a l c o u l d be d e t e c t e d  gram.  in the  i n the methyl-lysine  amounts o f H6, a s was r e p o r t e d ( 43 ) f r o m b i r d  [ *C] l a b e l ll  tamination  i n H6 c o u l d  by a n o t h e r  Similarly  o f t h e chromato-  h i s t o n e H3 was v e r y Since  erythrocytes. represent  Alternatively,  low l e v e l s  [ ^C]methylated molecule 1  Here, contamination likely  of only  forN -methyl-histidine  H2B showed n e g l i g i b l e  upon a n a l y s i s .  o f con-  ( e . g . H3, H 4 ) .  amounts o f m e t h y l - l y s i n e s with  s m a l l amounts o f  (see F i g . 3 ) .  H3, H4 a n d p o s s i b l y H6, a n d H2B c o n t a i n e d  lysines,  they  residues  i n t h e p r o t e i n were  I.  regions  f r o m H6  T h i s m i g h t b e due t o r a d i o a c t i v e l a b e l l i n g  H i a n d H5  resi-  pH 6.5 e l e c t r o p h o r e s i s ,  1  hydrolysates  Histone  o n l y mono- ar.d d i m e t h y l - l y s i n e , 0.43:0.27  s m a l l amounts o f [ ^C] b a s i c amino a c i d  trace  por-  were e x a m i n e d  f u r t h e r t o l o c a l i z e which  methyllysyl  methylated.  Sites of i n v i v o Methylation  o f L y s y l Residues  i n Testis  Histones (.a) The  Histone  H3  NH2-terminal o f t h i s  histone  i s not blocked,  so t h a t  -38-  it  was  feasible  to  methyl-labelled ically of  stable  t^C]  histone  to  the  This  C a n d i d o and  Dixon  In  14,  Fig.  For  As  methyl-lysines  hence of  ) who and  and  the  butane f r a c t i o n s number o f  and  (22  18  6,  III.  t e c h n i q u e was  t e s t i s h i s t o n e H3,  r e s i d u e s 9,  a u t o m a t e d Edman d e g r a d a t i o n s on  sequencing procedures,  radioactivity  localized.  of  perform  successfully  sequenced localized  from the  from a  for  a  semi-logarithmic  C a n d i d o and  Dixon  ).  d u c i b l e peak a t  cpm  was  for  also  4,  lysines  lysine  36,  recovery of  lysyl  of  a  against  plot  of  (Fig.  at  H3.  y i e l d of  93%,  y i e l d of  7)  as  27,  three A  alanyl  described  9 and  least  cpm  subtracted,  and  sites  small  (4,000 cpm  repetitive  by  one of  reprowhen  above a background o f  t h i s number o f [^C]  the  1  1,000  93%  chloro-  [ ^C]methyl l a b e l l e d  i n h i s t o n e H3.  yield)  d e t e c t e d , but after  residues  i n the  is plotted  lysines  indicating  repetitive  good r e s o l u t i o n The  major peaks a t  lysine  e-N-methylation of  corrected  (22  be  by  1  repetitive  degradations  m i n o r peak a t  25  b a c k g r o u n d r a d i o a c t i v i t y was  number o f  two  first  recovered  r e s i d u e s vs  There are  the  applied  [ "*C] a c e t a t e a t  sequenator  t h e n v a l u e s were c o r r e c t e d determined  positions  23.  radioactivity  the  the  chem-  methyl groups c o u l d  d e g r a d a t i o n s p e r f o r m e d on  each v a l u e ,  are  t^C]  yield  1,200  precludes  degradations.  r a d i o a c t i v i t y was  not  complete  -39-  T  NO. OF DEGRADATIONS  FIG. 6. A u t o m a t e d d e g r a d a t i o n o f [ C ] m e t h y l l a b e l l e d t e s t i s h i s t o n e H3. 12 mg o f h i s t o n e H3 (500,000 cpm) was s e q u e n c e d i n a Beckman 890 p r o t e i n s e q u e n c e r , u s i n g v o l a t i l e r e a g e n t s (122). R a d i o a c t i v i t y r e c o v e r e d a t each d e g r a d a t i v e step has been c o r r e c t e d f o r a r e p e t i t i v e y i e l d o f 93%, b a s e d on r e c o v e r y of a l a n y l r e s i d u e s , 1 l o g B, where Y i s t h e r e N - 1 p e t i t i v e y i e l d , N i s t h e number o f d e g r a d a t i o n s and B i s t h e r e c o v e r y o f amino a c i d a t s t e p N. So f o r Y = 93%, a t r e s i d u e 9 l o g (0.93) = 1 l o g B a n d B = 0.55. T h e r a d i o a c t i v i t y 9 - 1 a t s t e p 9 i s t h e r e f o r e c o r r e c t e d by a f a c t o r o f 1 . 0.55 1 4  Y  =  -40-  F I G . 7. Recovery o f a l a n i n e d u r i n g automated s e q u e n t i a l degr a d a t i o n o f h i s t o n e H 3 . The l o g a r i t h m o f t h e r e l a t i v e y i e l d o f a l a n i n e , p r e s e n t i n t h e s e q u e n c e a t r e s i d u e s 1,7,15,21,24,25, i s p l o t t e d a g a i n s t number o f d e g r a d a t i o n p e r f o r m e d . The N H 2 t e r m i n a l a l a n i n e i s t a k e n a s 100% r e l a t i v e y i e l d . Since Log  the  slope  Y = Trt—r M~ 1 ^ B ~ A -  B  o f t h e above p l o t ,  /  repetitive yield A, B a r e y i e l d s o f r e s i d u e s a f t e r N, o r N „ d e g r a d a t i o n s A B ^ l o g B/A v s N - N , g i v e s l o g Y. A  Y  =  n  a  -41-  from t h e sequencer. fined  To c o n f i r m  that methylation  t o the N-terminus o f the molecule,  180,000 cpm o f i n c o r p o r a t e d  [  llf  Sephadex G-50, a l m o s t radioactive atographed  peptide  50 mg o f H3  with  C ] m e t h y l g r o u p s , was c l e a v e d  w i t h N - b r o m o - s u c c i n i m i d e i n 50% a c e t i c When t h e r e s u l t i n g  o f H3 i s c o n -  acid.  p e p t i d e s were c h r o m a t o g r a p h e d o n  a l l [ ^ C ] i n c o r p o r a t i o n was i n a  single  1  NBS-1  ( F i g u r e 8) w h i c h , when r e c h r o m -  o n S e p h a d e x G-50 o r c a r b o x y m e t h y l c e l l u l o s e h a d  an  amino a c i d  composition  40  amino a c i d s e x p e c t e d  corresponding  t o the N-terminal  from an N - b r o m o s u c c i n i m i d e  cleavage  a t r e s i d u e 41, w h i c h i s a t y r o s i n e i n t h e h i s t o n e H3 o f various  species  (17-21).  c o u l d be a c c o u n t e d fractions  this  trypsin  confirmed  N-terminal  determined  by i s o l a t i n g  by a u t o m a t i c  labelled  p e p t i d e , NBS-1.  Peptide  tryptic  sequencing peptides  Following maleylation,  a n d c h r o m a t o g r a p h y on S e p h -  p e p t i d e s B l , B2 a n d B3 were i s o l a t e d B3 c o r r e s p o n d s  i s approximately  internal  s i d e o f the peak).  ( F i g . 9) and o n Whatman 3 MM p a p e r , t h r e e  radioactive  and  sites  ( F i g . 8,  of incorporation into  digestion, demaleylation  adex G-10  material  ( F i g . 8, f r a c t i o n s o n e i t h e r  methylation  were t h e n of  f o r as u n d i g e s t e d  20-30) o r low l e v e l s  methionine The  The r e s t o f t h e r a d i o a c t i v i t y  major  (Table I I I ) .  t o r e s i d u e s 27-40 f o r h i s t o n e H3  half-methylated  a t r e s i d u e 27, predom-  F I G . 8. Sephadex G-50 c h r o m a t o g r a p h y o f N - b r o m o s u c c i n i m i d e d e r i v e d p e p t i d e s o f [ ^ C ] m e t h y l l a b e l l e d h i s t o n e H3. 50 mg o f h i s t o n e H3 was r e a c t e d w i t h N - b r o m o s u c c i n i m i d e i n 50% a c e t i c a c i d , and t h e r e s u l t i n g p e p t i d e s were l y o p h i l i z e d and s e p a r a t e d on a Sephadex G-50 column i n 25% a c e t i c a c i d . P r o t e i n concent r a t i o n was m o n i t o r e d by r e a c t i o n w i t h n i n h y d r i n and m e a s u r e ment o f t h e a b s o r b a n c e a t 570 nm.  -43-  20  40  60  Fraction No., 2 m l  F I G . 9. Sephadex G-10 c h r o m a t o g r a p h y o f H3 t r y p t i c p e p t i d e s . [ ^ C ] m e t h y l l a b e l l e d p e p t i d e NBS-1, d e r i v e d f r o m H3 by N-bromos u c c i n i m i d e d i g e s t i o n , was m a l e y l a t e d a n d d i g e s t e d w i t h t r y p s i n . The r e s u l t i n g p e p t i d e s were f r a c t i o n a t e d on a 1.5 x 90 cm Sephadex G-10 c o l u m n , i n 10% a c e t i c a c i d . P e p t i d e was m o n i t o r e d by t h e n i n h y d r i n r e a c t i o n a t 570 nm.  -44-  TABLE I I I  [^C] methyl l a b e l l e d p e p t i d e s d e r i v e d from h i s t o n e H3  Peptide  N-terminal residue  Position  in  H i s t o n e H3 Sequence residues # 3  e-N - m e t h y l - l y s i n e s per p e p t i d e mono-  NBS-l B-l  b  c  di-  tri-  alanine  1-40  0.49  0.61  0.18  lysine  9-17  0.09  0.13  0.24  B-2  C  lysine  9-17  0.29  0.45  0.06  B-3  C  lysine  27-4 0  0.22  0.24  0.02  P e p t i d e s were p l a c e d by amino a c i d composition, N - t e r m i n a l a n a l y s i s and homology w i t h the h i s t o n e H3 o f o t h e r s p e c i e s (10,11,13,15,16). D e r i v e d from h i s t o n e H3 by d i g e s t i o n w i t h N-bromosuccinimide. Only h a l f (75,000 cpm) o f the p e p t i d e was m a l e y l a t e d and trypsin digested. D e r i v e d by t r y p t i c d i g e s t i o n o f m a l e y l a t e d NBS-1.  inantly  as e q u a l p r o p o r t i o n s o f t h e mono- a n d d i m e t h y l - l y s i n e  d e r i v a t i v e s ( T a b l e I I I ) . P e p t i d e s B l a n d B2, r e c o v e r e d i n approximately  e q u a l amounts, b o t h  correspond  to residues  9-17  i n h i s t o n e H3 ( T a b l e I I I ) . B l , h o w e v e r , h a s a l o w e r m o b i l i t y than  B2 on p a p e r c h r o m a t o g r a p h y  acid:water Bl  o r upon e l e c t r o p h o r e s i s a t pH 6.5,  has one l e s s p o s i t i v e  differs  i n butanol:pyridine:acetic  from  ( 15 , 22 ) .  B2 t h e n ,  charge  i n being  than  B2  suggesting  (131).  B l probably  acetylated at lysine  B l i s a l s o much l e s s m e t h y l a t e d  that  than  14  B2 b u t  a p p a r e n t l y c o n t a i n s most o f t h e e - N - t r i m e t h y l l y s i n e p r e s e n t i n h i s t o n e H3. in  The m e t h y l a t i o n  sites  o f H3 a r e s u m m a r i z e d  F i g . 14.  (b) As  Histone noted  methyl-lysine  H2B  i n section  I , t h e r e was some a p p a r e n t  i n c o r p o r a t i o n i n t o h i s t o n e H2B.  l y s i n e s w h i c h c o u l d be m e t h y l a t e d  i n this  Was l i k e  ( F i g . 10) g a v e a p r o f i l e t h a t f o r H3  been p r e s e n t  i n t h e H2B  p o o l i n g o f samples from  (c)  Histone  Histone  H6.  C]  There a r e  of this  of radioactivity  (at positions  ll4  protein at positions  5, 9, 10, 13, 14 e t c . , b u t a u t o m a t e d s e q u e n c i n g histone  [  which  4, 9, 27), w h i c h must  sample as a c o n t a m i n a n t  have  following  t h e B i o - G e l P-10 c o l u m n i n F i g . 3.  H6 This trout-specific  h i s t o n e showed a s m a l l  amount o f i n c o r p o r a t i o n a s [ "*C]-methyl amino a c i d , a n d 1  -46-  3r  No. of  Degradations  F I G . 10. A u t o m a t e d s e q u e n c e a n a l y s i s o f [ C ] m e t h y l l a b e l l e d t e s t i s h i s t o n e H2B. 7 mg o f t e s t i s H2B (40,000 cpm) was s e q u e n t i a l l y d e g r a d e d i n a Beckman 890 p r o t e i n s e q u e n c e r , u s i n q v o l a t i l e r e a g e n t s (122) . l l f  -47-  3  5  c o n t a i n s a sequence around  lysine  w h i c h i s homologous t o the  sequence m e t h y l a t e d  at positions automated labelled  9 and  27.  sequencing h i s t o n e H6  fourth degradation, up  For  arg - l y s -ser  reasons,  of the N-terminal  we  at lysine  Furthermore,  no  4.  sharp  as n o t e d  The group  Histone  procedures;  thus  from  such  which a f t e r  t h e amino a c i d leased tide  after  radio-  I  methylation calf  ( b ) , t h e r e were analysis.  instead.  r a d i o a c t i v e p e a k was  a Sephadex  obtained  ( F i g . 12)  arginine  3 Edman d e g r a d a t i o n s  proved  t o be  (1.02).  as was  L y s i n e 20  is  a  (1.16),  I n F i g . 13  radioactivity  are presented.  20,  G-25  ( F i g . 11),  t o r e s i d u e s 20-23 o f t h e p r o t e i n ,  occurring at lysine  [^C]  peptides  lysine + methyl-lysine  (1.00),  (12 ) .  acetyl  sequencing  When t h e  s e q u e n c e o f t h e p e p t i d e and  thymus h i s t o n e H4  an  peptides of maleylated  were a n a l y z e d  leucine  corresponds  the  peak o f r e l e a s e d in section  paper chromatography  (1.10),  r e l e a s e d on  i s u n a v a i l a b l e f o r automated  single peptide of composition valine  1  attempts  a d i g e s t were c h r o m a t o g r a p h e d on  column, a s i n g l e  [ "*C] m e t h y l  p r o t e i n i s b l o c k e d by  therefore, tryptic  m e t h y l h i s t o n e H4  that  H4  NH2-terminus o f t h i s  (12 ) and  expected  H3  However, s e v e r a l  no d e t e c t a b l e m e t h y l - l y s i n e s u p o n amino a c i d Cd)  (31 ),  i n histone  region of  w o u l d show r a d i o a c t i v i t y  t o r e s i d u e 25 y i e l d e d  activity.  these  4,  4  This  pep-  with  determined  re-  for  approximately  -48-  Fraction  No.,  10m/  F I G . 11. Sephadex G-25 c h r o m a t o g r a p h y o f t r y p t i c p e p t i d e s o f m a l e y l a t e d , [ ' " c i m e t h y l l a b e l l e d h i s t o n e H4. 70 mg o f [ C ] m e t h y l l a b e l l e d h i s t o n e H4 c o n t a i n i n g 150,000 cpm, was m a l e y l a t e d , d i g e s t e d w i t h t r y p s i n and c h r o m a t o g r a p h e d on a Sephadex G-2 5 c o l u m n i n 25% a c e t i c a c i d . P r o t e i n was m o n i t o r e d by t h e n i n h y d r i n r e a c t i o n a t 570 nm. I 4  -49-  solvent front  C5  C4  C3  C2  Cl  origin  FIG. 12. Paper chromatography o f t r y p t i c p e p t i d e s o f m a l e y l a t e d H4. F o l l o w i n g Sephadex G-25 f r a c t i o n a t i o n o f H4 t r y p t i c p e p t i d e s ( F i g . 1 1 ) , the [ ^ C ] m e t h y l l a b e l l e d peak was p o o l e d , c o n c e n t r a t e d and chromatographed on 3MM paper. The r a d i o a c t i v e p e p t i d e C5, was l o c a t e d by a u t o r a d i o g r a p h y . The o t h e r nonr a d i o a c t i v e p e p t i d e s C1-C4, not v i s i b l e i n the a u t o r a d i o g r a m , were l o c a t e d by n i n h y d r i n s t a i n i n g .  -50-  Me  Lys Val Leu Arg  Is  4  o o  b3  o  i  I  CJ  O  1 2 3 4 RESIDUE NUMBER  F I G . 13. S e q u e n c e a n a l y s i s o f [ C]methyl l a b e l l e d h i s t o n e H4. M a n u a l Edman d e g r a d a t i o n s were p e r f o r m e d on t h e s i n g l e [ C ] methyl l a b e l l e d p e p t i d e d e r i v e d from t r y p t i c d i g e s t i o n o f h i s t o n e H4. A l i q u o t s o f t h e r e l e a s e d PTH d e r i v a t i v e s were c o u n t e d i n Bray's s o l u t i o n . R a d i o a c t i v i t y r e l e a s e d i s p l o t t e d under t h e amino a c i d , i d e n t i f i e d by t h e s u b t r a c t i v e Edman p r o c e d u r e . 1U  -51-  70% methylated, being 1.6:1. The  the r a t i o s o f e-N-mono- t o d i m e t h y l - l y s i n e  There was no d e t e c t a b l e  trimethyl-lysine.  sequence c o n t a i n i n g t h e m e t h y l a t i o n s i t e i n t e s t i s  H4 i s shown i n F i g . 14. III.  (a)  H i s t o n e M e t h y l a t i o n i n the D i f f e r e n t  Developing T r o u t Having  established that s p e c i f i c l y s y l residues of h i s -  f o r o t h e r organisms  - as had been d e s c r i b e d  ( 12, 17, 21,100) - we were next  e s t e d i n the r e l a t i o n s h i p  inter-  of t h i s histone methylation t o the  spermatogenesis.  L o u i e and Dixon were a b l e t o s e p a r a t e , on bovine albumin  from  Testis  tones H3 and H4 were methylated  events o f  C e l l Types  gradients, different  testis cell  t h e i r sequence o f development a s : spermatocytes  ->• e a r l y ,  types and d e l i n e a t e  spermatogonia  l a t e spermatids  serum  1 ° , 2°  mature sperm.  Bio-  c h e m i c a l l y , DNA and h i s t o n e s y n t h e s i s stopped a t t h e spermatid  s t a g e , f o l l o w e d by the replacement  protamines  i n this c e l l  type  Was t h e r e a c o r r e l a t i o n  o f the h i s t o n e s by  (93 ) . between h i s t o n e m e t h y l a t i o n and  these c e l l types & b i o c h e m i c a l events?  To examine t h i s ques-  t i o n , a t e s t i s was o b t a i n e d 45 days a f t e r tions with p i t u i t a r y incubated w i t h the c e l l  extract.  A cell  [methyl- H]methionine 3  the s t a r t o f i n j e c -  suspension was made, and [ C ] l y s i n e , and 1 U  types were separated on a 1-3% bovine  serum  albumin  -52-  H3  Me Me Ac Ala-ArgThr Lys Glx Thr-Ala-Arg-Lys (Ser)4 9 Ac  Ac  14  18  Thr Gly Gly Lys Ala Pro Arg -Lys-Glx-LeuMe AlaThr-LysAlaAla-Arg-Lys -Ser-Ala-Pro 23 27 Ac  Ac  Ac  Ac  Ac-Ser-Gly Arg-Gly Lys Gly-Gly-Lys-Gly-Leu-Gly-Lys 5  12  8  Me Ac Gly-Gly Ala-Lys Arg-His-Arg- Lys-Val-Leu-Arg — 16  7  2o  F I G . 14. The s i t e s o f a c e t y l a t i o n a s d e t e r m i n e d by C a n d i d o and D i x o n ( 2 2 , 3 6 , 4 0 ) , and m e t h y l a t i o n o f t r o u t t e s t i s h i s t o n e s H3 and H4.  -53-  gradient each  ( 93,117).  fraction,  TCA-tungstate Figure  cells  shows t h a t  and  3  Sv).  macromolecules, proteins.  acid  extraction  electrophoresis  DNA  and  fold  on  histone methyl  I,  into  specifically cell  i n F i g . 15). directly  the  into  cell  into  types  The  his-  (pooled  results  starch  analysis  larger  of  gel  slots,  of the h i s t o n e s  diploid  spermatocytes  (pool b ) , which are s t i l l ( c f [ C]lysine 1  a per c e l l  basis).  incorporation  rather  than as  The  This  in  spermatid  histone methylation accounts  As  noted  (20-30  f o r the  i n the u n f r a c t i o n a t e d  [ H]methyl incorporation 3  active  incorporation),  h  shows much l o w e r  a l l of the  lysine  spermatids  TCA-tungstate  of methionine  i n which spermatids predominate.  almost  methyl  that  stem c e l l s  ( p o o l c)  lower,  file,  t h a n do  f o r most o f t h e h i s t o n e m e t h y l a t i o n .  fraction  f a r more  from m e t h y l a t i o n o f o t h e r  radioactivity  histone synthesis  account  basis,  (3.5  16.  i s apparent and  incorporate  f o r the d i f f e r e n t  o f the c e l l s  shown i n F i g .  ( p o o l a)  derive  fractions  and  determined.  spermatocytes  of t h i s  Therefore, incorporation determined  was  and  represents methylation of histone  or i n c o r p o r a t i o n  the g r a d i e n t  It  on a p e r c e l l  some c o u l d  from  are  Sv)  For  a hemocytometer,  larger  (2.8  incorporation  residues;  t o n e s was  the  However, o n l y a p o r t i o n  precipitable lysyl  on  radioactivity  stem c e l l s  [methyl- H]methionine (1.5  o f 7 m l were c o l l e c t e d .  were c o u n t e d  precipitable  15  mm/hr = Sv)  Fractions  cell  pro-  in section  o c c u r s as  i n t e r n a l methionine;  low  hence,  e-Nthe  T———i  FRACTION  1  r  NO. (7.0ml)  FIG. 15. I n c o r p o r a t i o n o f L - [ m e t h y l - H ] m e t h i o n i n e and L - f ^ C ] l y s i n e i n t o d i f f e r e n t c e l l types from t r o u t t e s t i s . A testis c e l l s u s p e n s i o n , from a f i s h 45 days a f t e r the s t a r t o f maturat i o n , was i n c u b a t e d w i t h [ H]methionine and [ C]lysine a t 16°C f o r 2 hours and was f r a c t i o n a t e d a t u n i t g r a v i t y on a 1-3% bovine serum albumin g r a d i e n t (93,117). C e l l s were counted on a hemocytometer, and r a d i o a c t i v i t y was determined on an a l i q u o t of each f r a c t i o n on a m i l l i p o r e f i l t e r , as d e s c r i b e d i n " M a t e r i a l s and Methods". Numbers over the arrows r e f e r t o sedimentation v e l o c i t y (Sv) i n m i l l i m e t e r s per hour. 3  3  lk  -55-  W1A  H1.H2A.H3  H»  4II 9'  Cells  9  I  i  •  Fl  i  r  :  1M  I 5  ii.  i  '•  b  c  J  1  0  2  °  •5  (  a o  «  60  o  DISTANCE FROM  SO  100  ORIGIN, CM  FIG. 16. S y n t h e s i s and m e t h y l a t i o n o f h i s t o n e s i n the d i f f e r e n t c e l l types from t r o u t t e s t i s . C e l l s from r e g i o n s a, b and c i n F i g u r e 15 were pooled, and [ C]lysine and [ H]methyl i n c o r p o r a t i o n i n t o h i s t o n e s was determined by s t a r c h g e l e l e c t r o p h o r e s i s as d e s c r i b e d i n " M a t e r i a l s and Methods". The h i s t o n e bands a r e i d e n t i f i e d i n F i g u r e 16a; P-H2A r e f e r s t o phosphorylated H2A. lh  3  -56-  m e t h y l i n c o r p o r a t i o n on  the  starch gel  i s solely into his-  11+  t o n e s H3 all  and  the  whereas  [  C]lysine  i s incorporated  into  histones.  (b) We  H4,  T u r n o v e r o f m e t h y l g r o u p s on were n e x t  i n t e r e s t e d i n the  groups i n t r o u t t e s t i s methylation  cells.  turnover  There are  i s a- s t a b l e m o d i f i c a t i o n  servations  show i t t o be  groups t u r n i n g  histone  the  cell  of  residues  histone  reports  that  methyl histone  (108), w h i l e o t h e r  a dynamic p r o c e s s ,  over during  lysyl  cycle  with  ob-  methyl  (100,107).  The  Ik results of  e x p e r i m e n t s on  population  of  Figure  i s shown i n F i g u r e  15)  trout testis  methyl turnover radioactive viability L-[  up  to  label.  as  [  Parallel  i n t o 5%  insoluble material  cells 17.  T h e r e i s no the  incubations  the  ability  of  The  could  methyl i n c o r p o r a t i o n cytes IV.  versus  account  cells  r e s u l t s presented  tone m e t h y l a t i o n occurs mainly cytes,  cells  cell  incorporate tungstate turn-  differential  differences in  l a r g e r stem c e l l s  histone  and  spermato-  spermatids.  K i n e t i c s o f H i s t o n e H4 The  to  lack of detectable  f o r the  between t h e  of  demonstrated  t r i c h l o r o a c e t i c a c i d - 0.2%  ( F i g . 18).  in  detectable  removal  o v e r o f m e t h y l g r o u p s makes i t u n l i k e l y t h a t methyl turnover  w i t h a mixed  ( s i m i l a r to t h a t used  9 hours f o l l o w i n g  m e a s u r e d by  H]arginine  C]methyl turnover  Methylation in section  is a relatively  i n the  stable modification,  large diploid  which s t i l l  are  III indicate that  stem c e l l s  synthesizing  DNA  his-  which  and  spermato-  and  histones.  -57-  4  6  8  Hours after Chase FIG. 17. Turnover o f [^C] methyl groups i n t r o u t t e s t i s h i s tones. A t e s t i s c e l l suspension (5 x 1 0 c e l l s ) was i n c u b a t e d a t 16°C f o r 2 hours w i t h 100 yCi/ml o f D L - [ H ] l y s i n e and 50 yCi/ml o f L - [ m e t h y l - "*C]methionine. The c e l l s were then c o l l e c t e d by c e n t r i f u g a t i o n , resuspended i n 8 ml o f i s o t o p e - f r e e medium, and a l i q u o t s c o n t a i n i n g 5 x 1 0 • c e l l s were removed a t i n t e r v a l s and c o l l e c t e d on M i l l i p o r e g l a s s f i b e r f i l t e r s . R a d i o a c t i v i t y i n the h i s t o n e s was determined by s t a r c h g e l e l e c t r o p h o r e s i s ; [ C ] m e t h y l and [ H] l y s i n e i n c o r p o r a t i o n were integrated over the h i s t o n e r e g i o n and the r a t i o o f [ *C]methyl t o [ H ] l y s i n e i n c o r p o r a t i o n was then c a l c u l a t e d f o r each time sample. 8  3  1  1  llf  3  1!  3  -58-  K  E CL u  CO  o I  z  F I G . 18. Incorporation of [ H]arginine into t e s t i s nuclei with time-cell viability. A f t e r i n c u b a t i o n of t e s t i s c e l l s f o r 2 h o u r s W i t h [ H ] l y s i n e and [ m e t h y l - C ] m e t h i o n i n e (see F i g . 1 7 ) , an a l i q u o t o f c e l l s was f u r t h e r i n c u b a t e d w i t h 50 y C i / m l o f L-[ H]arginine. A t v a r y i n g .times, t o t a l r a d i o a c t i v i t y i n c e l l n u c l e i was d e t e r m i n e d by p r e c i p i t a t i o n w i t h T C A - t u n g s t a t e on glass fiber f i l t e r s . 3  3  3  1 4  -59-  We next examined t h e r e l a t i o n s h i p o f t h i s to  methylation  the s y n t h e s i s , a c e t y l a t i o n and p h o s p h o r y l a t i o n  H4.  H4 can be separated  10 d i f f e r e n t s p e c i e s H4 w i t h  on u r e a - l a c t a t e s t a r c h g e l s i n t o  (61), c o n s i s t i n g o f unphosphorylated  0 to 4 e-N-acetyl-lysines  phosphorylated  of histone  H4 w i t h  (Ao t o A ) and slower mono4  0 t o 4 a c e t a t e groups  (PiAo t o PiAit),  as i n F i g u r e 19. When Louie and Dixon l a b e l l e d h i s t o n e H4 w i t h for  i n c r e a s i n g p e r i o d s o f time  synthesized,  (84) , they  [ H ] l y s i n e - l a b e l l e d H4 was f i r s t d e t e c t a b l e 3  position  (A2).  only  Only  ( a f t e r 16 hours) d i d [ H ] l y s i n e l a b e l appear i n 3  the unmodified species  3  found t h a t newly  as t h e unphosphorylated, d i a c e t y l a t e d s p e c i e s much l a t e r  [ H]lysine  (Ao, A i ) and monophosphorylated  ( P i A , P1A1) 0  ( F i g . 2 0 ) . In o t h e r words, they c o u l d r e l a t e the ( i . e . degree o f a c e t y l a t i o n ) o f h i s t o n e H4 r a d i o -  a c t i v i t y on a s t a r c h g e l t o the time a f t e r s y n t h e s i s o f t h e h i s t o n e H4. for  On t h e b a s i s o f these r e s u l t s , they  postulated  newly s y n t h e s i z e d h i s t o n e H4 an o b l i g a t o r y a c e t y l a t i o n  and d e a c e t y l a t i o n c y c l e , which would be necessary b i n d i n g t o DNA  for correct  (84).  As t h e r e i s evidence  that histone methylation  occurs  l a t e i n t h e c e l l c y c l e a f t e r h i s t o n e and DNA s y n t h e s i s (100, 132),  i t was o f i n t e r e s t t o see whether  [ *C]methyl l a b e l l e d ll  h i s t o n e H4 appeared a t t h e A2 p o s i t i o n c h a r a c t e r i s t i c o f newly s y n t h e s i z e d h i s t o n e , o r a t the unmodified monophosphorylated H4.  ( A , A ) and 0  x  ( P i A , P1A1) p o s i t i o n s o f " o l d " h i s t o n e 0  F o r t h i s purpose, c e l l  suspensions were l a b e l l e d  with  -60-  FIG. 19. The a c e t y l a t e d and phosphorylated s p e c i e s o f h i s t o n e H4, separated by s t a r c h g e l e l e c t r o p h o r e s i s . Histone H4, p r e pared from t r o u t t e s t i s by a c i d e x t r a c t i o n , and carboxymethylc e l l u l o s e and B i o - G e l P-10 chromatography, was separated on a u r e a - l a c t a t e s t a r c h g e l i n t o the m o d i f i e d s p e c i e s . The bands are l a b e l l e d a c c o r d i n g t o Sung and Dixon (61); s u b s c r i p t numbers r e f e r t o number o f e-N-acetyl (A) or phosphoryl (P) groups per h i s t o n e H4 molecule. Bands were v i s u a l i z e d by s t a i n i n g the g e l w i t h Amido Black.  -61-  105 DISTANCE  12  ia5 FROM  15 ORIGIN, CM  FIG. 20. [ H ] l y s i n e i n c o r p o r a t i o n i n t o h i s t o n e H4 w i t h t i m e , as d e s c r i b e d by L o u i e and Dixon (84) . T e s t i s c e l l s were i n c u b a t e d w i t h [ H ] l y s i n e and a t v a r y i n g time i n t e r v a l s H4 was prepared. For longer incubations (>12 h o u r s ) , [ H ] l y s i n e was i n j e c t e d i n t e r p e r i t o n e a l l y i n t o l i v e t r o u t a t time = 0. The m o d i f i e d s p e c i e s o f H4 were r e s o l v e d , and [ H ] l y s i n e i n c o r p o r a t i o n was d e t e r m i n e d w i t h u r e a - l a c t a t e s t a r c h g e l s . 3  3  3  3  -62-  (methylwas It  1  1  * C ) m e t h i o n i n e f o r up  purified  and  H4,  [  ( t = 30 min) (PiAo, at  electrophoresed  i s c l e a r from F i g u r e  histone  of  methyllysine  activity  H4  t h a t u n l i k e newly  a t the unmodified  H4  [^Cjmethyl b u t as  i n the A  2  monophosphorylated  label  (less  i s incorporated  n o t as  e-N-  species.  i n t o newly  [  x l |  synthesized  levels  of radio-  With continued C]methyl  label  incubation i n t h e mono-  i n c r e a s e s , a t r a n s i t i o n which  obtained  f r o m 16 h r t o 1-3  by L o u i e days  H4  amount  1  (new)  of histone  A small  [ ''C] m e t h i o n i n e  species  the r e s u l t s  immediately  (Ao, A i ) and  and c o u l d a c c o u n t f o r t h e low  phosphorylated  t o n e H4,  appears  synthesis.  (Figure 21), the p r o p o r t i o n of  lels  H4  synthesized  positions characteristic  16 h o u r s a f t e r  10%)  histone  21,  H4  on u r e a - l a c t a t e s t a r c h g e l s .  C]methyl l a b e l l e d  PiAi) regions,  least  than  1 I +  t o 12 h o u r s , t h e n h i s t o n e  and  after  Dixon  (84)  synthesis.  paral-  for his-  -63-  FIG. 21. [ C ] m e t h y l i n c o r p o r a t i o n i n t o h i s t o n e H4 as a funct i o n o f time. Trout t e s t i s c e l l s were incubated w i t h L-[methyl " C ] m e t h i o n i n e , a t v a r y i n g time i n t e r v a l s a l i q u o t s were r e moved, and h i s t o n e H4 was prepared. The m o d i f i e d s p e c i e s o f h i s t o n e H4 were r e s o l v e d and [ ^ C ] m e t h y l i n c o r p o r a t i o n was determined, f o r each time p o i n t , w i t h u r e a - l a c t a t e s t a r c h g e l s . 14  1  1  -64-  DISCUSSION T h e r e a p p e a r t o be no g r e a t histone testis  methylation cells  (lysine and  20) o f h i s t o n e  thymus and o t h e r [  1 4  between t e r m i n a l l y d i f f e r e n t i a t i n g  and o t h e r  27) o f h i s t o n e  tissues studied.  H3 a r e i d e n t i c a l  tissues  (15-20), except  from c h i c k e n , The  low l e v e l s  H6, w i t h o u t protein,  shark,  could r e f l e c t  36 i n h i s t o n e  and m o l l u s c  with  H3.  (lysine  histone i n the  of the p u r i f i e d  histone  H3 o r H4.  mated p r o t e i n s e q u e n c i n g d i d n o t however g i v e  4)  (lysine 36).  amounts o f e - N - m e t h y l - l y s i n e s some c o n t a m i n a t i o n  These  study o f  incorporation into  t o n e H6 b y t r a c e amounts o f l a b e l l e d  radioactivity  for calf  labelling  H3 f r o m a c y c a d  sea urchin  of [^Clmethyl  detectable  (lysines 9  (21) i n a c o m p a r a t i v e  sequences o f h i s t o n e  2  site  reported  that  trout  the d e t e c t i o n o f a d d i t i o n a l minor  m i n o r s i t e s were a l s o o b s e r v e d  and  t o those  a t . l y s i n e 4 and p o s s i b l y l y s i n e  NH -terminal  The s i n g l e  H4 and t h e two m a j o r s i t e s  C ] m e t h y l groups allowed  sites  differences i n the s i t e s of  his-  Auto-  a profile of  r e l e a s e d which i s c h a r a c t e r i s t i c  of histone  H3.  1 4  Alternatively,  [  C]methyl i n c o r p o r a t i o n could  amounts o f m e t h y l a t i o n histidine  i n avian  similar  erythrocyte  t o that observed histone  to histone  among m e t h y l a t e d  H4, H3) and - A l a - A r g - L y s - S e r -  sites of histone methylated  sequences:  H3).  However, h i s t o n e  trace  f o r 3-methyl-  H5 b y G e r s h e y e t a l . ( 4 3 ) .  DeLange, S m i t h a n d c o - w o r k e r s h a v e n o t e d ilarities  reflect  (9,10) t h e s i m -  -x-Arg-Lys-x(for both  (common  major  H6 i s n o t s i g n i f i c a n t l y  i n t h e sequence - A r g - L y s - S e r around  lysine  4.  This  -65-  suggests t h a t e i t h e r ( i ) r e c o g n i t i o n by the methylase r e q u i r e s s t r u c t u r a l f e a t u r e s a d d i t i o n a l t o those peptide-Arg-Lys-Ser;  or  i n the  ( i i ) the l o c a t i o n or s t r u c t u r e o f  h i s t o n e H6 makes i t i n a c c e s s i b l e to m e t h y l a t i o n . and  tri-  Purification  s t u d i e s on the s u b s t r a t e s p e c i f i c i t y o f the h i s t o n e methyl-  ase a c t i v i t y p r e s e n t  i n t r o u t t e s t i s n u c l e i might r e s o l v e t h i s  question. There i s as y e t no i n f o r m a t i o n on the f u n c t i o n o f H6.  T h i s minor  h i s t o n e H2B  (1% o f t o t a l ) , t r o u t - s p e c i f i c h i s t o n e  i n s i z e , and  l y s i n e to a r g i n i n e r a t i o  histone resembles  (19),  but  i s l i k e H i i n i t s e x t r a c t a b i l i t y from chromatin, i t s h i g h content  of l y s i n e , a l a n i n e and p r o l i n e , and  modifications ated nor  (H6 can be p h o s p h o r y l a t e d ,  i t s postsynthetic  but i s n e i t h e r a c e t y l -  methylated)"*".  None o f the o t h e r h i s t o n e s c o n t a i n e d  s i g n i f i c a n t amounts  of m e t h y l - l y s i n e s except h i s t o n e H2B, which was inated with  H3.  Histones  H3  and H4  are h i g h l y conserved i n amino a c i d  sequence and m e t h y l a t i o n  s i t e s i n d i f f e r e n t species, yet  are d i f f e r e n c e s i n the e x t e n t o f m e t h y l a t i o n sites  (the most dramatic  l y s i n e i n pea  l i k e l y contam-  there  of l y s i n e s a t  those  b e i n g the complete absence of methyl-  histone H4).  The  r e s u l t a n t heterogeneity  of  h i s t o n e molecules w i t h r e s p e c t t o number of methyl groups, as p o i n t e d out by A l l f r e y  (3), makes i t d i f f i c u l t  generalized or universal function for histone  ^ B.M.  Honda, unpublished  results  to a s s i g n a methylation.  -66-  Histone Methylation i n D i f f e r e n t are  incubated with  Cell  Types.  [methyl- H]methionine  and  3  When t e s t i s separated  g r a v i t y , methyl i n c o r p o r a t i o n i n t o histones occurs inantly and  i n the  16).  l a r g e spermatocytes  This methylation  probably  has  no  role  stem c e l l s and  are  undergoing  still cell  the condensation  from  the d i p l o i d  actively division  (93).  of chromatin  spermatocytes and  Groups.  It  (93),  (78). and  histones that  to m i t o s i s The  (100,108).  lack of detectable  ( F i g . 18)  makes i t u n l i k e l y for either  with respect to  larger  stem c e l l s  and  sperm-  spermatids.  i s possible that a very  occurs over  prot-  ( i i ) the d i f f e r e n c e s i n h i s t o n e  m e t h y l i n c o r p o r a t i o n between t h e atocytes versus  so  i n these processes, or i n  the h e t e r o g e n e i t y of h i s t o n e molecules  number o f m e t h y l g r o u p s o r  and  o f h i s t o n e s by  t h a t d i f f e r e n t i a l methyl turnover could account (i)  ( F i g . 15  I t i s p o s s i b l e then  prior  turnover of h i s t o n e methyl groups  one  predom-  spermatids  s y n t h e s i z i n g DNA  some r o l e  of Histone Methyl  at  require histone acetylation  spermatids,  h i s t o n e m e t h y l a t i o n has  Turnover  i s absent  stem c e l l s  i n the displacement  amine, a p r o c e s s w h i c h may U n l i k e the  and  cells  a cell  slow t u r n o v e r o f methyl  c y c l e , which f o r t r o u t  much l o n g e r t h a n  the time  testis  i s 6-7  of the experiment.  complete turnover of methyl groups d u r i n g a c e l l  groups days  However,  cycle  appears  unlikely. A methyl turnover estimated  by  r a t e o f 2%  per  B o r u n e t a l . (107), b u t  hour i n Hela this  cells  e s t i m a t e was  was  based  -67-  14  on r a t i o s o f  3  C to  about t h e mean r a t e .  •  H i n c o r p o r a t i o n w h i c h were w i d e l y s c a t t e r e d Moreover, t h e p u t a t i v e p r o t e i n "demethyl-  ase" o f P a i k and Kim (109), w h i c h m i g h t be r e s p o n s i b l e f o r m e t h y l group t u r n o v e r , has n o t been shown t o a c t on c h r o m a t i n a s s o c i a t e d h i s t o n e s , and i t s s u b s t r a t e s and p r o d u c t s have n o t y e t been c l e a r l y  identified.  In t r o u t t e s t i s c e l l s then, histone methylation i s a relatively stable modification. K i n e t i c s of Histone  H4 M e t h y l a t i o n .  L o u i e and D i x o n (84)  demonstrated t h a t newly s y n t h e s i z e d H4 underwent an o b l i g a t o r y r a p i d a c e t y l a t i o n t o t h e d i a c e t y l a t e d form A2, f o l l o w e d by a s l o w e r a c e t y l a t i o n , p h o s p h o r y l a t i o n , and d e a c e t y l a t i o n pathway. The  r e s u l t s o f [^C] m e t h y l l a b e l l i n g o f h i s t o n e H4 w i t h  time a l l o w us t o p l a c e H4 m e t h y l a t i o n i n t o t h i s scheme o f H4 metabolism.  Since the behaviour  of [  1I#  C]methyl l a b e l l e d  his-  tone H4 ( F i g . 21) p a r a l l e l s t h a t o f h i s t o n e H4 some 16 h o u r s t o 1-3 days a f t e r s y n t h e s i s , t h i s s u g g e s t s s y n t h e t i c events  t o us t h a t p o s t -  i n h i s t o n e H4 m e t a b o l i s m c o n s i s t o f a c e t y l a -  t i o n and d e a c e t y l a t i o n (up t o 12-16 h o u r s ) , f o l l o w e d by m e t h y l a t i o n and p h o s p h o r y l a t i o n . I t i s u n l i k e l y t h a t m e t h y l a t i o n o c c u r s on a s m a l l p o o l o f h i s t o n e H4 b e h a v i n g d i f f e r e n t l y from t h e m a j o r i t y . a pool  Such  (which, f o r example might bypass a c e t y l a t i o n ) would be  a t most a few p e r c e n t o f t h e t o t a l , j u d g i n g from t h e r e s u l t s o f L o u i e and Dixon ( 8 4 ) , whereas m e t h y l a t i o n o c c u r s on a t  -68-  least  70%  o f h i s t o n e H4  Furthermore, species of  the  results  and  and  late  prior If  course  h o u r s t o 1-3  correlate well with  Paik  occurs  16  Kim  (100),  i n the c e l l  to chromatin  the  in this  of methyl  shows a good t e m p o r a l  h i s t o n e H4  and  shift  molecules  label  tissue  to  phosphorylated  correspondence days a f t e r  (section I I ) .  with  the  synthesis.  behaviour These  t h e work o f T i d w e l l e t a l .  (132)  showing t h a t h i s t o n e  methylation  cycle,  histone synthesis  after  condensation  same a c e t y l a t i o n ,  DNA  and  and m i t o s i s .  deacetylation, methylation  i s a p p l i c a b l e t o h i s t o n e H3,  then  time  the d i f f e r e n c e s  in  j.  e x t e n t o f m e t h y l a t i o n and  acetylation  b e t w e e n H3  peptides  Bl  and  composition  see T a b l e  III) might  be  explained.  B2  recently H3  (with i d e n t i c a l  molecules,  w h e r e a s p e p t i d e B2  molecules.  The  answer emerges. tion  sites  would d e r i v e from  presence  of r e l a t i v e l y  and  28;  From t h e o b s e r v a t i o n s — 9,27)  sites"*"  o f h i s t o n e H3  (also inferred  high 'levels  to i n t e r p r e t ,  clear  are adjacent r e f . 38)  to  methylathe  at serines  i s associated with  cell  phorylation  c o i n c i d e n t l y with, or a f t e r methylation  B.M.  Honda, u n p u b l i s h e d  and  No  however.  the l a r g e r d i v i d i n g occurs  types  histone  (i) t h a t the  from  ( i i ) that histone methylation  from  o l d e r , more  function of histone methylation?  (lysines  phosphorylation  derived  undermethylated  e-N-trimethyl-lysine i n Bl i s d i f f i c u l t What i s t h e  10  ( T a b l e I I I ) w o u l d be  s y n t h e s i z e d , a c e t y l a t e d and  methylated of  Peptide B l  results  ( i i i ) t h a t h i s t o n e H4  phos—  -69-  it  i s possible that histone methylation  to phosphorylation mitosis.  and  i s a necessary  subsequent chromatin  This methylation  may  prelude  condensation  i n v o l v e changes  in  charge,  conformation  or h y d r o p h o b i c i t y of  actions with  a histone phosphokinase or other molecules.  H3  and  H4  interact with  each o t h e r  and  the h i s t o n e , a l l o w i n g  (32-34) i n t e t r a m e r s  interSince to  g i v e a s u b u n i t - l i k e s t r u c t u r e t o c h r o m a t i n - ( s e e P a r t B, thesis), tural  methylation  changes  of these  i n chromatin  and  r e g u l a t i o n of the  ifications these  should  cellular  the  struc-  division.  isolation, characterization  enzymes r e s p o n s i b l e f o r h i s t o n e mod-  clarify  events.  2 h i s t o n e s might a l s o induce  required for c e l l  F u r t h e r e x p e r i m e n t s on  this  the  role  of histone methylation  in  PART B:  CHROMATIN SUBUNIT STRUCTURE -70-  INTRODUCTION The  structure  and f u n c t i o n o f c h r o m a t i n — t h a t  of  DNA, h i s t o n e s ,  in  t h e c e l l n u c l e u s — has l o n g been a m y s t e r y .  Part A of this present  non-histone proteins  thesis,  the histones  taining gene  chromatin  activity.  (NHP)  vary  structure  Unlike  species  a review,  see  (136).  the n e c e s s i t y  (133-135.)  brane  (137) The  tissue  (134). of  the non-histone  Reports  histones,  weight.  o n NHP s t o i c h i o m e t r y  case the authors  f o r NHP t o r e g u l a t e large  amounts  gene  (134)  amounts  (138). These p r o t e i n s  and have been  in genetically  synthesis,  rather,  o f NHP a s p o s s i b l e mem-  contaminants  specificity  activity;  exhibit  described  active  phosphorylation or  tissues  turnover  NHP h a s i m p l i c a t e d them i n t h e i n d u c t i o n o f  (134),  (139-141).  i n molecular  do n o t d i s -  The i n c r e a s e d  activity  proteins  In the l a t t e r  i n increased  specific  It  a s f o r example has a l s o  confer  reconstituted  of  p o p u l a t i o n w i t h many  daltons  f u n c t i o n s o f NHP a r e u n c l e a r .  present  control  t o o n l y 3%  or cytoplasmic  and species  i n main-  o f e q u a l w e i g h t w i t h DNA (135)  they q u e s t i o n reported  as  t o be i m p o r t a n t  the histones,  proteins  There are 5 major  and p o s s i b l y " c o a r s e "  up t o 1 0 0 , 0 0 0  from r e p o r t s  by w e i g h t pute  thought  o f RNA  As noted i n  are small basic  o f chromatin are a heterogeneous  different (For  a n d s m a l l amounts  i n e q u a l w e i g h t w i t h t h e DNA.  classes of these p r o t e i n s ,  complex  i n response  been r e p o r t e d  tissue-specific  chromatins  (142) .  that  to steroid  gene  hormones  t h e NHP, n o t  transcription  activity  T h e NHP may a l s o  to  regulate  -71-  cell NHP in  division;  t h e s y n t h e s i s and p h o s p h o r y l a t i o n o f  at different  stages of the c e l l  support of t h i s  hypothesis  be n u c l e a r enzymes of  cycle  (143).  (134) o r p o s s i b l y  different  have been d e s c r i b e d  Finally,  some NHP  structural  must  components  chromatin. In  what  in  chromatin?  of  chromatin  Wilkins spaced they  kind  of structure  Until  recently,  came f r o m  interpreted  proteins  (145).  diffraction  from c h r o m a t i n  chromatin  regular,  a s a DNA  random  helix  multiples  of a unit  p r o t e i n s were  but r a t h e r  length.  arranged  protecting  found  coated  of proteins  with The  on  that  an  c l e a v e d t h e DNA,  to discrete  T h i s suggested  not  fragments,  t o them  that the  i n an o r d e r e d , r e p e t i t i v e manner a l o n g  t h e DNA  (147) t h a t  nuclei  (146) n o t e d  from n u c l e a s e d i g e s t i o n .  treatment o f i n t a c t  rat liver  w i t h t h e endogenous n u c l e a s e o r c o m m e r c i a l l y c o c c a l n u c l e a s e c l e a v e d 85% o f t h e DNA long or multiples  thereof.  Louie  r e s u l t s w i t h polyoma n u c l e o p r o t e i n . isolate,  this  o r s u p e r h e l i x (144).  or other —  in rat liver  randomly t o a l l s i z e s ,  pairs  From  remained u n c e r t a i n .  endogenous n u c l e a s e  then  model  regularly  fibers.  double  T h e n i n 1973, H e w i s h a n d B u r g o y n e  t h e DNA,  assembled  studies of  They o b s e r v e d  a n d wound i n a l a r g e r c o i l  arrangement — t h e DNA  rings  and p r o t e i n s  t h e most w i d e l y a c c e p t e d  the X-ray  (144) and o t h e r s diffraction  a r e DNA  nuclei  available  t o fragments  micro-  200  (148) o b t a i n e d  Noll  from n u c l e a s e - t r e a t e d n u c l e i ,  Noll  (147) c o u l d  intact  s u b u n i t s " , c o n t a i n i n g h i s t o n e s and n o n - h i s t o n e  11. 2S  base similar  also  "chromatin  proteins  -72-  a t t a c h e d t o s t r e t c h e s of DNA and coworkers (149)  200 base p a i r s l o n g .  Van  had e a r l i e r d e s c r i b e d d i s c r e t e  r e s u l t i n g from m i c r o c o c c a l n u c l e a s e d i g e s t i o n o f  particles  chromatin.  From t h e o r e t i c a l c o n s i d e r a t i o n s , f o r each h i s t o n e H i  molecule  t h e r e c o u l d be two each o f H2A,  H2B,  weight o f DNA  to a length of roughly  which corresponds  base p a i r s as observed.  H3 and H4,  Holde  E l e c t r o n micrographs  l i k e r e g i o n s spaced a l o n g chromatin  (150)  and an  equal 200  showing bead-  supported such a  p i c t u r e o f s u b u n i t s o f n u c l e a s e - r e s i s t a n t , protein-bound a l t e r n a t i n g w i t h n u c l e a s e - s e n s i t i v e , DNA Romberg then extended  DNA  s t r u c t u r e (152)  base p a i r s and two  histone I ) .  spacer r e g i o n s .  such r e s u l t s , along w i t h h i s own  on c r o s s l i n k e d h i s t o n e oligomers chromatin  DNA  data  (151), i n t o a theory o f  based on a r e p e a t i n g u n i t o f  200  of each o f the h i s t o n e s (except f o r  A s i m i l a r model was  proposed  by Van Holde and  coworkers (153) . N o l l a l s o observed t h a t when chromatin  i n s i t u was d i -  g e s t e d w i t h DNase I , a r e g u l a r s e r i e s o f s i n g l e - s t r a n d e d DNA fragments,  m u l t i p l e s o f 10 bases l o n g , were o b t a i n e d  H i s work s t r o n g l y suggested  t h a t DNA  on the o u t s i d e of the chromatin  i s a c c e s s i b l e t o DNase,  s u b u n i t , the s t r u c t u r e o f  which c o n t a i n s some i n t e r n a l r e p e t i t i v e elements. DNA  fragments,  ed by Van Holde  (154).  Different  s m a l l e r than 200 base p a i r s l o n g , had been r e p o r t (149), A x e l e t a l . (155), and Weintraub & coworkers  (156) when i s o l a t e d chromatin,  as opposed t o i n t a c t n u c l e i , were  -73-  treated with micrococcal nuclease. t o some r e g u l a r r e p e a t "specific arise  s t r u c t u r e f o r chromatin,  c o n t a c t s between p r o t e i n  from  Such r e s u l t s  and n u c l e i c  posed N - t e r m i n i  of a t r y p s i n - r e s i s t a n t  i t was d i f f i c u l t  ducts  to those At  t h e time  trout  study  of chromatin  free  treatment  then,  structure of  since:  chromatin  (i) large quantities  s t r u c t u r e when t h e h i s t o n e s  d u r i n g sperm d e v e l o p m e n t ; and  modifications of histones i n trout  of nuclei  thesis,  from  trout  chromatin  subunits t o those  This  further  200 b a s e p a i r  we  i s a good t i s s u e .for t h e  testis  supports  at early  DNA  of the  nuclease (histone)  f r a g m e n t s and 1.1 S  reported i n other studies t h e model o f e u k a r y o t i c  (146-148).  chromatin  l o n g segments o f DNA c o v e r e d w i t h p r o t e i n s .  T h e s e s u b u n i t s w o u l d be s e p a r a t e d by n u c l e a s e - s e n s i t i v e spacer  DNA.  (iii)  testis.  i t i s shown t h a t  stages o f development g i v e s s i m i l a r  data  pro-  of cytoplasmic contamination are  changes i n chromatin  In P a r t B o f t h i s  as  digestion  s u b u n i t s p r o v i d e a model s u b s t r a t e f o r t h e study  enzymatic  (156),  ( i i ) t h e s u b u n i t s r e p r e s e n t one a p p r o a c h t o  a r e r e p l a c e d by p r o t a m i n e the  the subunit  subunits  relatively  (156).  (147).  Trout t e s t i s  chromatin  which  h i s t o n e complex  i n v e s t i g a t i o n was s t a r t e d  i n examining  prepared;  studying  this  testis.  o f such  easily  acid  on  o f DNA by t h e e x -  these nuclease  o b t a i n e d by N o l l  were i n t e r e s t e d from  to relate  pointed  based  s t r u c t u r a l properties of the histones"  T h i s c o u l d have i n v o l v e d t h e c r o s s l i n k i n g  but  also  -74-  Testis (meiotic  c o n s i s t i n g predominantly of e a r l y  tissue,  similar yields stage  testis  containing mainly  o f DNA  nucleohistone)  f r a g m e n t s and  ( p r o t a m i n e has  replaced  11S the  l i s subunits.  subunits. histones)  g i v e s no  DNA  reflects  l a r g e d i f f e r e n c e s i n s t r u c t u r e between  amine and  fragments or  spermatids  This  gives Later however,  presumably nucleoprot-  nucleohistone.  Finally,  the  chromatin subunits  composition are  more r e c e n t f i n d i n g s .  and  reported,  some p r o p e r t i e s o f and  discussed  testis  i n terms  of  -75-  MATERIALS.AND METHODS I.  C h e m i c a l s and A b b r e v i a t i o n s (a)  Chemicals  A l l c h e m i c a l s o b t a i n e d c o m m e r c i a l l y were o f t h e h i g h e s t p u r i t y o r reagent grade.  S p e c i a l r e a g e n t s were o b t a i n e d as  follows:  (E.C. 1.11.1.6) from W o r t h i n g t o n ;  Bovine c a t a l a s e  m i c r o c o c c a l n u c l e a s e (E.C. 3.1.4.7) from Sigma; a c r y l a m i d e from Matheson,  Coleman and B e l l ; TEMED  (N,N,N*,N'-tetramethyl-  e t h y l e n e d i a m i n e ) from C a n a l I n d u s t r i a l Corp; N, N'-methy1ene— b i s a c r y l a m i d e and " S t a i n s a l l " (b) EDTA: SDS:  from Eastman Kodak.  Abbreviations ethylenediamine-tetraacetic sodium  Stainsall:  acid  dodecyl s u l f a t e l-ethyl-2-[3-(1-ethylnaphth  [1,2a]-thiazolin-  2-ylidene)-2-methylpropenyl]naphtho[1,2a]-thiazolium TMKS b u f f e r : KC1  (50 mM, pH 7 . 4 ) , M g C l  2  (1 mM),  (25 mM), and s u c r o s e (0.25 M ) . TMKSM b u f f e r :  :i.  Tris-HCl  bromide  TMKS b u f f e r made 15 mM i n 2 - m e r c a p t o e t h a n o l  P r e p a r a t i o n and N u c l e a s e D i g e s t i o n o f N u c l e i o r I s o l a t e d Chromatin N a t u r a l l y maturing t r o u t testes  ( o b t a i n e d from Sun V a l l e y  T r o u t Farm, M i s s i o n , B.C.) were v i g o r o u s l y homogenized i n TMKSM b u f f e r , and n u c l e i were p e l l e t e d a t 1,000 g f o r 10 m i n . A f t e r two more washings w i t h t h e same b u f f e r , 5 - 10 x 1 0  8  -76-  n u c l e i / r a l were i n c u b a t e d a t 37°C f o r 1-10 units/ml  In l a t e r p r e p a r a t i o n s o f n u c l e i ,  o m i t t e d from the b u f f e r s .  t h r e e times i n 10 mM  0.1 III.  mM  T r i s pH  o f DNA/ml) was  A260  2  M NaCl,  8.0.  performed  mM  2-mercaptoethanol  Chromatin was  washing n u c l e i f u r t h e r w i t h 0.15  (20  A 6o  o f m i c r o c o c c a l n u c l e a s e i n TMKSM b u f f e r made 1  in CaCl2. was  min w i t h 500  prepared  20 mM  Digestion of  by  EDTA, and  then  chromatin  a t 37°C i n 10 mM  Tris,  C a C l , w i t h 300 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e . 2  D e t e r m i n a t i o n o f Developmental Stages  (histone:protamine  r a t i o ) of Testes N u c l e i prepared as above were e x t r a c t e d w i t h 0.4  N H2SCH  and b a s i c p r o t e i n s were p r e c i p i t a t e d w i t h 3 volumes o f e t h a n o l a t -20°C.  P r o t e i n s were e l e c t r o p h o r e s e d on urea-15% p o l y -  a c r y l a m i d e g e l s (157)  s t a i n e d w i t h Coomassie Blue and  a t 600 nm w i t h a G i l f o r d spectrophotometer and Methods, s e c t i o n V I I I  scanned  [see M a t e r i a l s  (b) f o r a c r y l a m i d e g e l e l e c t r o -  phoresis] . IV.  P r e p a r a t i o n of "Chromatin S u b u n i t s " from D i g e s t e d N u c l e i A f t e r treatment w i t h m i c r o c o c c a l n u c l e a s e , n u c l e i were  c o l l e c t e d a t 1000  g, resuspended  and spun a t 12,000 g f o r 30 min (147).  The  v i g o r o u s l y i n 0.2  mM  as f i r s t d e s c r i b e d by  s u p e r n a t a n t , c o n t a i n i n g 10-200 A 6 o / m l o f  s u b u n i t s , was  2  then l a y e r e d on a 10-30% sucrose-0.2  mM  EDTA, Noll chromatin EDTA  -77-  gradient  (12 ml) a n d s p u n a t  a Beckman S W 4 0 - T i r o t o r .  In rpm  tube,  o f 0.4  o f t h e g r a d i e n t w i t h 60%  earlier  experiments,  collected  catalase,  and  were  16S r e g i o n s For  After  s u b u n i t s were  a digest  (a)  were d i g e s t e d  i n 5 ml o f  This  gave  approximately  the c e n t r i f u g a t i o n at  o f DNA F r a g m e n t s  Isolation  n u c l e i were  Bovine  or chromatin,  phenol extraction  f o r t h e 11S  nuclei  TMKS-CaCl2 nuclease. resuspended 80-100  12,000 g ,  30 m i n .  nuclease-treated  o r from i s o l a t e d chromatin s u b u n i t s , (147).  S a m p l e s i n 1% S D S , M N a C l ,  20  DNA was p r e c i p i -  from t h e r e s u l t i n g aqueous phase w i t h 2 volumes o f -20°C.  A260/  o f DNA f r a g m e n t s  mM EDTA were e x t r a c t e d t w i c e w i t h p h e n o l .  ethanol at  sep-  Produced by N u c l e a s e D i g e s t i o n  DNA was p r e p a r e d f r o m m i c r o c o c c a l  tated  frac-  sedimenting i n a  c o n t a i n i n g p r e d o m i n a n t l y monomers,  ml o f s u b u n i t s a f t e r  by  and twenty drop  8 m i n , w i t h 500 u n i t s / m l o f m i c r o c o c c a l  Analysis  25,000  respectively.  d i g e s t i o n and c e n t r i f u g a t i o n ,  nuclei  spun a t  found t o be c o n v e n i e n t markers  2 m l o f 0 . 2 mM E D T A .  V.  collected  sucrose.  3-galactosidase,  f r o m 2 grams o f t e s t i s buffer,  m l were  from t h e tube bottom by s i p h o n i n g .  and E . c o l i  arate tube,  hours i n  f o l l o w e d by upward d i s -  f o r 18 h r i n a Beckman SW27 r o t o r ,  tions  in  rpm f o r 12-14  Fractions  by p u n c t u r i n g t h e g r a d i e n t placement  3 6,000  -78-  (b)  Q u a n t i t a t i o n of  DNA  In t h i s procedure d e s c r i b e d by Burton (10-300 yg/ml) were made 0.5 a t 70°C f o r 15 min. r e a c t e d w i t h 0.67  samples  N i n p e r c h l o r i c a c i d and  A f t e r c o o l i n g , a 0.33  ml of diphenylamine  amine i n 100 ml a c e t i c a c i d p l u s 1.5 acetaldehyde had been f r e s h l y added acetaldehyde per 20 ml o f r e a g e n t ) . 30°C f o r 16-20  (158), DNA  h r , and the absorbance  heated  ml a l i q u o t  reagent  (1.5 g d i p h e n y l -  ml H2SOO  t o which  (0.1 ml o f 1.6% C o l o u r was  aqueous  developed  a t 565 nm was  a g a i n s t a b l a n k , and s t a n d a r d c a l f thymus DNA  was  at  monitored  samples o f known  concentration. (c)  Non-denaturing  phoresis of  2.5%  polyacrylamide gel e l e c t r o -  DNA  In t h i s technique d e s c r i b e d by Loening  (159), the f o l l o w -  i n g volumes o f s t o c k s o l u t i o n s : - 5 ml o f lOx c o n c e n t r a t e d TEA b u f f e r 2 mM  (TEA b u f f e r = 0.04  EDTA, 0.02  solution  M T r i s - a c e t i c a c i d pH  M sodium a c e t a t e ) , 6.25  ml o f 20%  ( a c r y l a m i d e : N,N'-methylenebisacrylamide,  7.8,  acrylamide 19:1)  -  were combined w i t h 38 ml o f water and deaerated under vacuum. Then 40 mg  of ammonium p e r s u l f a t e i n 0.7  ml o f H 2 O ,  y l of TEMED (N,N,N',N'-tetramethylethylenediamine) added, and the a c r y l a m i d e was i n 7 cm  tubes.  and  40  were  p o l y m e r i z e d under i s o b u t a n o l  , -79-  Samples o f 5-100 yg o f DNA i n 10% s u c r o s e , 0.005% bromop h e n o l b l u e were e l e c t r o p h o r e s e d i n TEA b u f f e r a t 4°C f o r 1-lh  hours a t 5 m i l l i a m p e r e s / g e l .  in Stainsall  (0.005% i n 50% formamide), t h e n d e s t a i n e d i n  w a t e r by b r i e f exposure t o l i g h t . a t 550 nm i n a G i l f o r d (d)  G e l s were s t a i n e d o v e r n i g h t  Denaturing  G e l s were t h e n scanned  spectrophotometer.  99% formamide, 6% p o l y a c r y l a m i d e g e l separation  o f DNA F o r t h e s e g e l s d e s c r i b e d by Staynov e t a l . (160), 99% formamide i s s t i r r e d w i t h Dowex 50W-X8 (3 g/100 ml) f o r 1 h o u r , and i s t h e n f i l t e r e d and u s e d t h e same day. F o r a 6% acrylamide  (85:15) g e l , 2.04 g o f a c r y l a m i d e , 0.36 g o f N,N'-  methylenebisacrylamide  and 80 y l o f TEMED were d i s s o l v e d i n  40 m l o f formamide, w h i c h was t h e n f i l t e r e d .  Then 50 mg o f  ammonium p e r s u l f a t e , d i s s o l v e d i n 0.8 m l o f M sodium phosphate pH 7.0, was added t o make t h e g e l 20 mM i n phosphate and 0.12% ammonium p e r s u l f a t e .  A 15 x 15 x 0.15 cm s l a b g e l ,  s e a l e d a t t h e bottom w i t h p l a s t i c s p a c e r and v a s e l i n e , was t h e n poured and p o l y m e r i z e d . RNA and DNA samples were d i s s o l v e d i n formamide ( c o n t a i n i n g 20 mM sodium phosphate pH 7.0, 20% s u c r o s e , 0.005% bromop h e n o l b l u e ) , heated t o 70°C, and e l e c t r o p h o r e s e d i n formamide20 mM phosphate a t up t o 100 v o l t s , 10 m i l l i a m p e r e s f o r 12-16 hours.  G e l s were s t a i n e d i n S t a i n s a l l o v e r n i g h t and d e s t a i n e d  i n l i g h t , , as d e s c r i b e d above.  -80-  VI.  Sepharose  2B C h r o m a t o g r a p h y o f C h r o m a t i n  Chromatin  s u b u n i t s were p r e p a r e d  section  I I I ) i n 0.2 mM  40 cm S e p h a r o s e at  4 ° C i n 5 mM  VII.  Velocity  2B  (Materials  (Pharmacia)  Tris-HCl,  column, e q u i l i b r a t e d  0.2 mM  Sedimentation  on  i s applied  Beckman M o d e l E A n a l y t i c a l U l t r a c e n t r i f u g e ,  molecules ary  with  of molecules,  efficient  w r 2  from  the rotor  1  to dr • —  relationship  In ^  To  =  center; field  ^  i s then  2  strength.  Rearranging the  .  .  (t-to).  S 2 0  ,  w  water),  = {l-VPWo,w  2  n the solution  a v a l u e o f s,  b. S20/W  (s v a l u e a t  t h e equation used i s :  ^  where V i s t h e p a r t i a l and  T i n buffer  ;  gives the  From t h e s l o p e , w s , o f  the s value to a standard  20°C i n d i s t i l l e d  bound-  the v e l o c i t y ,  w s • d t , and i n t e g r a t i n g , w s  of  dr 1 S = v r . — 2 — , where r i s t h e at w r  ?  =  a t temperature correct  cleared  near  the sedimentation co-  a p l o t o f I n r / r o v s t - t o , one c a n c a l c u l a t e obtained  of the  the region  i s progressively  one c a n d e t e r m i n e  i s the centrifugal  equation,  cell  solu-  From t h e v e l o c i t y o f t h i s m o v i n g  o f the molecules,  displacement and  time.  Subunits  to a uniform  i n the sector-shaped  (top) o f the c e l l  and r u n  EDTA, pH 7.6.  Experiments  field  o f macromolecules  the meniscus  and Methods,  EDTA, a n d c h r o m a t o g r a p h e d o n a 2.5 x  When a c e n t r i f u g a l tion  Subunits  .  specific  viscosity.  s  volume,  p the solution  density  -81-  Samples o f s u b u n i t s i n 20 mM were sedimented c e l l a t 4°C.  Tris,  a t 36,000 o r 40,000 rpm  The moving boundary  c o n c e n t r a t i o n vs d i s t a n c e p r o f i l e  was  0.2 mM  EDTA, pH  i n a 1.61  cm  7.0  sector  monitored t o g i v e a  a t 15-20  min i n t e r v a l s i n  e i t h e r o f two ways as f o l l o w s ; (i)  Scanner o p t i c s .  c e n t r a t i o n o f 0.8 through the c e l l  A26o/ml.  UV l i g h t  (A = 260 nm)  a t a coni s passed  and darkens a p h o t o g r a p h i c p l a t e .  ance, which w i l l r i s e boundary,  Subunits are sedimented  from 0 a t the meniscus,  t o 0.8  Absorba t the  i s measured by d e n s i t o m e t r i c scanning o f the photo-  graphic p l a t e .  T h i s g i v e s an absorbance vs d i s t a n c e  profile  at each time p o i n t . (ii) A2 6 o / m l . fractive  Schlieren optics.  A t the moving boundary, index  (An)  parallel  t h e r e i s a change i n r e -  such t h a t p a r a l l e l  through i t are d e f l e c t e d trifuge,  Subunits are sedimented a t 60  light  l i g h t rays passing  (as i n a p r i s m ) .  In the u l t r a e e n -  i s passed through the c e l l ,  through a s e r i e s o f l e n s e s and a d i a g o n a l s l i t angle  and then  (inclined  at  0). L i g h t r a y s p a s s i n g above o r below the molecule  boundary  i n the c e l l are not d e f l e c t e d  ( A n = o ) , and g i v e a s t r a i g h t  l i n e on a p h o t o g r a p h i c p l a t e .  However, l i g h t r a y s p a s s i n g  through the boundary  are d e f l e c t e d  ( A n > o ) , and a r e d i s p l a c e d  sideways on the p h o t o g r a p h i c p l a t e , i n a d i s p l a c e m e n t p r o -  -82-  p o r t i o n a l t o An.  The  r e s u l t on the p h o t o g r a p h i c p l a t e a t  the p o s i t i o n o f t h e boundary, i s a peak w h i c h c o r r e s p o n d s mathematically  t o the f i r s t d e r i v a t i v e ( g ^ )  p r o f i l e o b t a i n a b l e w i t h scanner o p t i c s . subunits, 0 = VIII.  o f  t h e  c  Y_s r  In experiments w i t h  50°.  P r o t e i n Composition of Chromatin Subunits (a)  I s o l a t i o n o f p r o t e i n s from s u b u n i t s  o r whole c h r o m a t i n  S e v e r a l methods were used t o remove p r o t e i n s from  subunit  DNA. (i) i n SDS gel  A l i q u o t s o f c h r o m a t i n s u b u n i t s were made 1 t o  and  h e a t e d t o 100°C t o r e l e a s e p r o t e i n s p r i o r t o  electrophoresis.  S u b u n i t s were sometimes t r e a t e d  DNase I (5 yg/sample) p r i o r t o SDS clogging of polyacrylamide p r e c i p i t a t e d w i t h 10 mM min, 20%  5%  gels.  sucrose,  phoresis,  0.15  with  e x t r a c t i o n , to avoid ( i i ) S u b u n i t s were  M g C l , c o l l e c t e d a t 2,000 rpm 2  t h e n h e a t e d t o 100°C i n 0.1%  SDS  SDS,  10 mM  T r i s pH  M 2 - m e r c a p t o e t h a n o l b e f o r e SDS  DNA  first for  10  7.2,  gel electro-  ( i i i ) S u b u n i t s were p r e c i p i t a t e d w i t h M g C l , t h e n 2  p r o t e i n s were e x t r a c t e d w i t h 0.2  N  HC1.  N o n - h i s t o n e chromosomal p r o t e i n s were i s o l a t e d f r o m whole chromatin  [prepared  as i n M a t e r i a l s and Methods, s e c t i o n I I ]  as d e s c r i b e d by L e S t o u r g e o n and  Rusch (161) , by  sequential  t r e a t m e n t w i t h s a l t , a c i d , b u f f e r - s a t u r a t e d p h e n o l (pH and hot SDS.  A l t e r n a t i v e l y , c h r o m a t i n was  N H SOi| (removing h i s t o n e s ) , o r g a n i c 2  8.2)  extracted with  solvent  (removing  0.4  lipid)  -83-  and f i n a l l y w i t h 1% SDS,  0.14 M 2 - m e r c a p t o e t h a n o l , 10 mM  sodium phosphate pH 7.2, t o d i s s o c i a t e n o n - h i s t o n e essentially (b)  as d e s c r i b e d by E l g i n and Bonner  proteins,  (162).  Gel electrophoresis of proteins  Three systems were a p p l i e d t o c h r o m a t i n  proteins, the  f i r s t two b e i n g s p e c i f i c a l l y f o r b a s i c p r o t e i n s ( h i s t o n e s and  protamines). (i)  P r o t e i n samples were e l e c t r o p h o r e s e d on u r e a -  aluminum l a c t a t e s t a r c h g e l s as d e s c r i b e d by Sung and Smithies  (120) [see t h i s t h e s i s , P a r t I , M a t e r i a l s and  Methods, s e c t i o n I I ( b ) ] . (ii)  P r o t e i n samples were e l e c t r o p h o r e s e d on u r e a - 1 5 %  p o l y a c r y l a m i d e g e l s as d e s c r i b e d by Panyim and C h a l k l e y (157).  The f o l l o w i n g volumes o f s t o c k s o l u t i o n s : - 1 m l  TEMED s o l u t i o n (48 m l N KOH, 17.2 m l a c e t i c a c i d , 4 m l TEMED, 30 ml o f H 2 O ) , 2 m l a c r y l a m i d e  solution  (60 g a c r y l a m i d e ,  0.4 g N , N ' - m e t h y l e n e b i s a c r y l a m i d e i n 100 m l H 2 O ) and 5 m l persulfate-urea solution  (0.2% ammonium p e r s u l f a t e i n 10 M  urea) - were combined and p o l y m e r i z e d urea.  i n g e l t u b e s under 3 M  P r o t e i n (10-50 ug) was l o a d e d i n 6 M u r e a w i t h m e t h y l  green as a marker, and e l e c t r o p h o r e s e d i n 3 - a l a n i n e t r a y buffer  (31.2 g 3 - a l a n i n e , 8 ml a c e t i c a c i d d i l u t e d t o 1000  ml w i t h H 2 O ) a t 4-5 m i l l a m p e r e s  p e r g e l f o r 1-1% h o u r s .  G e l s were s t a i n e d o v e r n i g h t w i t h Coomassie B r i l l i a n t  Blue  -84-  ( S e r v a , Germany) i n m e t h a n o l : a c e t i c destained  i n the  (iii) Osborn  The (163)  0.1%  SDS  5%  were u s e d  polyacrylamide  ml  of II  in  2 ml  H2O)  0.5  M  bined with in  tubes  heated  and  15  10.0  o f 10%  SDS,  and  of  317  4.5  or  ml  f o r 4-5  freshly  g  follow-  acrylaH2O),  of  dissolved  M NaH P0^, 145  ml  2  o f H 0) 2  o f H 0,  - were com-  and  2  polymerized usually 7.2,  0.1% 2  at  SDS  10%  elecmade  7-8  G e l s were s t a i n e d w i t h  d e s t a i n e d as d e s c r i b e d a b o v e .  A b s o r b a n c e a t 230  c o n c e n t r a t i o n s show an A 2 3 0  Bonner e t a l . (164), and  0.2 0.4  nm.  Histone  absorbance, o f  standards  3.5  A230  per  at  vary-  1 mg/ml.  - 1 mg/ml.  Trichloroacetic  H2O  ml  ml  I I I w i t h H 0)  hours.  By  ml  ml  (22.2  i n 100  (0.1 M p h o s p h a t e ,  (i)  1  of I  The  0.002%-bromophenol b l u e , t h e n  stock s o l u t i o n  per tube  content.  s o d i u m p h o s p h a t e pH  Q u a n t i t a t i o n of p r o t e i n s  0.6  and  (28.25 ml  (c)  (ii)  g e l s o f Weber  P r o t e i n s a m p l e s were  Tris  in tray buffer  For protamines,  with  III  10 mM  C o o m a s s i e B l u e and  ing  of  isobutanol.  SDS,  dilution  milliamperes  ml  y l o f TEMED and  or g l y c e r o l  trophoresed 1:1  - 4.5  ammonium p e r s u l f a t e  10 ml  under  i n 1%  sucrose  by  (30 mg  Na2HPO4,  and  to analyse p r o t e i n  g IS^N'-methylenebisacrylamide  1.0  5:1:5,  same s o l v e n t .  i n g volumes o f s t o c k s o l u t i o n s : m i d e , 0.6  acid:water,  acid ml  ml  turbidity.  I n t h i s method  of  o f h i s t o n e sample i s c o m b i n e d o f 50%  trichloroacetic  acid  (3.3  M).  -85-  The  resulting  after  the  tone are (iii) mide o r  turbidity  r e a c t i o n was  i s monitored started.  done c o n c u r r e n t l y .  gel  scans.  gels,  areas under each scanned p r o t e i n  nm  solutions of  his-  yg/ml o f Urea-15%  stained with  histone. polyacryla-  Coomassie  s p e c t r o p h o t o m e t e r and  the  peak w e r e m e a s u r e d .  t o amount o f  t h o u g h c o r r e c t i o n s were n e c e s s a r y d i f f e r e n t molecular weight  min  0  SDS-5% p o l y a c r y l a m i d e  proportional  13-15  1 At, o - 107.5  From p o l y a c r y l a m i d e  was  400  Standard  B l u e , were scanned w i t h a G i l f o r d  binding  at  (bigger  Dye  protein present,  i n comparing proteins  al-  proteins  bind  more  of  dye).  RESULTS I.  C h a r a c t e r i z a t i o n o f DNA c o c c a l Nuclease I n 1973,  tion  o f DNA  sizes,  but  of a u n i t  Burgoyne  in rat liver  r a t h e r DNA  lengths  Noll  ly  400,  suggested  from  We  (147)  confirmed  development.  no  protamines) used  (a)  an  this  nuclease  digestion  by  These r e s u l t s  DNA  testis  fragments  such  digested  nucleohistone stage  ted with micrococcal nuclease, f r a g m e n t s when a n a l y z e d  of  strong-  DNA,  far  every  200  between.  the on  stages  testes i s similar  2.5%  i n the r a t  and liver  Noll.  nuclei  testis DNA  of  i n pro-  meiotic tissue,  B u r g o y n e , and  from  one  at early  p r o p e r t i e s to chromatin  H e w i s h and  DNA  i n looking at micrococcal  i n trout from  and  yielded  repetitive  (and c o n t a i n s l i t t l e  and  When e a r l y  crete  result  gave s i m i l a r  ordered  interested  Chromatin  composition  nuclei  this  with nuclease-sensitive regions  d i g e s t e d DNA  nuclease  fragments o f a l l  t h a t t h e a r r a n g e m e n t o f p r o t e i n s on  were f i r s t  nuclease  endogenous  e t c . base p a i r s .  b e i n g random, was  base p a i r s ,  tein  600  reported that diges-  f r a g m e n t s w h i c h were i n t e g r a l m u l t i p l e s  Nuclei treated with  200,  an  smear o f DNA  showed t h a t m i c r o c o c c a l n u c l e a s e patterns.  (146)  n u c l e i by  the expected  length.  Micro-  Digestion  H e w i s h and  produced, not  F r a g m e n t s P r o d u c e d by  nuclei  i s reduced  are  diges-  to  dis-  polyacrylamide  gels  -87-  (Fig. l a ) .  Extended d i g e s t i o n o f such n u c l e i c o n v e r t s  e r than 80% o f the DNA t o fragments c o r r e s p o n d i n g smallest u n i t length  (Fig. l b ) .  (from r a t l i v e r  w i t h the same 2.5% p o l y a c r y l a m i d e (147).  He p r o v i d e d  t o the  N o l l has c a l i b r a t e d t h e  m o b i l i t i e s o f such DNA fragments  DNA markers  great-  nuclei)  g e l system a g a i n s t  sequenced  good evidence t h a t t h e f r a g -  ments so produced a r e 205 ± 15 base p a i r s l o n g , and m u l t i p l e s (approximately  400, 600 e t c . base p a i r s ) t h e r e o f .  A plot of  l o g DNA band number vs square r o o t o f band m o b i l i t y f o r these fragments was l i n e a r  (147), where the s m a l l e s t fragment i s  band 1 (monomer), the next l a r g e s t i s band 2 (dimer)  etc.  A p l o t o f l o g DNA band number vs square r o o t o f m o b i l i t y i s a l s o l i n e a r f o r t h e DNA fragments from t r o u t t e s t i s ( F i g . 2), i n d i c a t i n g t h a t t h e l a r g e r DNA fragments a r e a p p r o x i m a t e l y i n t e g r a l m u l t i p l e s i n l e n g t h o f the s m a l l e s t DNA monomer piece.  We estimate  a single-stranded  l e n g t h o f roughly  30 n u c l e o t i d e s f o r t h i s t r o u t monomer DNA p i e c e , u s i n g acrylamide (160)  g e l e l e c t r o p h o r e s i s i n 99% formamide  against Drosophila  200 ± poly-  ( F i g . 3)  5S and tRNA markers, and based on  N o l l ' s c a r e f u l c a l i b r a t i o n o f RNA and DNA m o b i l i t i e s i n t h i s system  (154) ( F i g . 4) .  C l o s e r examination o f t h e s m a l l e s t t r o u t DNA fragment shows t h a t i t c o n s i s t s o f two s p e c i e s c o r r e s p o n d i n g monomer, 200 ± 30 base p a i r s , and a s m a l l e r , c l e a v e d monomer DNA, 175 + 25 base p a i r s  to  partially  ( F i g . 5a, bands 1  -88-  b  i  0  i  i  20  i  i  4.0  Distance,  i  cm.  6.0  FIG. 1. 2.5% p o l y a c r y l a m i d e g e l scans o f DNA: (a) from h i s tone s t a g e n u c l e i d i g e s t e d w i t h 100 A 2 6 0 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e , 3 min (b) from h i s t o n e s t a g e n u c l e i d i g e s t e d w i t h 600 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e , 6 min (c) from l a t e p r o t a m i n e stage n u c l e i d i g e s t e d w i t h 600 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e , 6 min. DNA was p r e p a r e d from n u c l e i by p h e n o l e x t r a c t i o n , and was e l e c t r o p h o r e s e d on L o e n i n g (159) 2.5% g e l s as d e s c r i b e d i n Methods. G e l s were s t a i n e d i n S t a i n s a l l , d e s t a i n e d i n l i g h t and scanned a t 550 nm i n a G i l f o r d spectrophotometer.  -89-  F I G . 2. 2.5% p o l y a c r y l a m i d e g e l s o f DNA f r a g m e n t s f r o m d i g e s ted n u c l e i . P l o t o f DNA band number a g a i n s t s q u a r e r o o t o f band m o b i l i t y . Data i s t a k e n from t h e g e l i n F i g u r e l a .  -90-  FIG. 3. D e n a t u r i n g 99% formamide, 6% p o l y a c r y l a m i d e g e l separation of DNA f r a g m e n t s . DNA, p r e p a r e d by p h e n o l e x t r a c t i o n from n u c l e i , was d i s s o l v e d i n b u f f e r e d formamide, h e a t e d t o 70°C and e l e c t r o p h o r e s e d on 6% g e l s as i n Methods, a c c o r d i n g t o t h e p r o cedure o f S t a y n o r e t a l . (160). G e l s were s t a i n e d i n S t a i n s a l l and l i g h t d e s t a i n e d .  5 Distance  10 migrated  F I G . 4. C a l i b r a t i o n o f m o b i l i t y v s s i n g l e s t r a n d e d l e n g t h f o r n u c l e i c a c i d s on d e n a t u r i n g 99% f o r m a m i d e , 6% p o l y a c r y l a m i d e g e l s a c c o r d i n g t o N o l l (154). The r e l a t i v e m o b i l i t i e s o f . D r o s o p h i l a 5S RNA f _ A ) , tRNA ( • ) , t e s t i s monomer ( [JIT ) a n d trimmed monomer (|o |) were d e t e r m i n e d f r o m a f o r m a m i d e g e l and superimposed on N o l l ' s c a l i b r a t i o n c u r v e . The b o x e s a r e meant t o i n d i c a t e t h e e r r o r s p o s s i b l e (=15%) i n s u c h a n e s t i m a t i o n .  -92-  and  la).  N o l l has estimated  a l e n g t h o f 170 + 10 base p a i r s  f o r t h i s c l e a v e d monomer (147) from r a t l i v e r n u c l e i . s m a l l e r monomer probably  This  d e r i v e s , as suggested by N o l l  from e n d o n u c l e o l y t i c cleavage  (147),  i n p a r t o f the 200 base p a i r s  p r o t e c t e d by p r o t e i n , r e s u l t i n g i n a "trimming" o r removal o f a more nuclease  a c c e s s i b l e 30-35 base p a i r spacer  region  between 170 base p a i r long p r o t e i n - p r o t e c t e d r e g i o n s . On 2.5% g e l s which have been o v e r l o a d e d  w i t h monomer  DNA, two fragments even s m a l l e r than 170 base p a i r s appear ( F i g . 6) but i n extremely low y i e l d compared w i t h the unf r a c t i o n a t e d DNA sample  (not overloaded) shown i n F i g . 6.  These two s m a l l fragments again probably endonuclease cleavages  represent  internal  a t l e s s a c c e s s i b l e s i t e s i n the pro-  t e i n - p r o t e c t e d DNA r e g i o n s . (b)  DNA fragments from d i g e s t e d chromatin  When i s o l a t e d chromatin, as opposed t o i n t a c t i s digested with micrococcal nuclease  nuclei,  f o r 1-10 minutes, the  same DNA fragments can be i s o l a t e d and c h a r a c t e r i z e d on 2.5% polyacrylamide  gels  ( F i g . 5b) o r as s i n g l e - s t r a n d e d fragments  on 99% formamide, 6% p o l y a c r y l a m i d e 5b)  gels.  C l e a r l y here ( F i g .  t h e s m a l l DNA fragment c o n s i s t s o f 2 s p e c i e s , monomer  (band 1) and "trimmed" monomer (band l a ) . The nuclease  r e c o v e r y o f such d i s c r e t e fragments from m i c r o c o c c a l d i g e s t s f u r t h e r support  the model  (14 6-154) t h a t  -93-  0.8  0.4  O io b 3 2 II  1 1a  BAND NO.  0.8  0.4  3.0  0 Distance  migrated,  6.0 cm.  F I G . 5. C h a r a c t e r i z a t i o n o f DNA f r a g m e n t s (a) f r o m i n t a c t n u c l e i a n d (b) f r o m i s o l a t e d c h r o m a t i n , d i g e s t e d w i t h m i c r o coccal nuclease. I n t a c t n u c l e i (a) o r i s o l a t e d c h r o m a t i n (b) were d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e under i d e n t i c a l c o n d i t i o n s a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . DNA was p r e p a r e d b y p h e n o l e x t r a c t i o n and e t h a n o l p r e c i p i t a t i o n , a n d a n a l y s e d on 2.5% p o l y a c r y l a m i d e g e l s . G e l s were s t a i n e d i n " S t a i n s a l l " , a n d s c a n n e d a t 550 nm i n a G i l f o r d s p e c t r o p h o t o meter.  -94-  monomer, bands 1,1a minor  bands  FIG. 6. 2.5% p o l y a c r y l a m i d e g e l o f D N A e x t r a c t e d from chrom a t i n s u b u n i t monomers. D N A was phenol e x t r a c t e d , e l e c t r o phoresed as f o r F i g . 1 then s t a i n e d w i t h S t a i n s a l l and des t a i n e d i n l i g h t . Note the 2 D N A bands s m a l l e r than monomer band 1.  -95-  chromatin  (in s i t u i . e . i n nuclei, or isolated) consists  l a r g e l y o f 200 base p a i r l o n g segments o f DNA, c o v e r e d  with  p r o t e i n s , p a r t o f which i n c l u d e s a s m a l l spacer r e g i o n o f DNA (30-35 base p a i r s ) between s u b u n i t s , w h i c h i s a c c e s s i b l e to nuclease d i g e s t i o n . II.  P r e p a r a t i o n o f I s o l a t e d Chromatin  Subunits  I n a d d i t i o n t o c h a r a c t e r i z i n g DNA fragments produced by m i c r o c o c c a l n u c l e a s e d i g e s t i o n , N o l l succeeded i n s e p a r a t i n g chromatin  from n u c l e a s e - t r e a t e d n u c l e i i n t o 11.2S  " s u b u n i t monomers" ( c o n t a i n i n g chromosomal p r o t e i n s on 200 base p a i r s o f DNA) and l a r g e r o l i g o m e r s  (147).  This  s e c t i o n d e t a i l s t h e p r e p a r a t i o n and s i z e d e t e r m i n a t i o n o f chromatin (a)  s u b u n i t s from t r o u t t e s t i s . Sucrose g r a d i e n t p r e p a r a t i o n o f chromatin  subunits  C h r o m a t i n was i s o l a t e d i n 0.2 mM EDTA from n u c l e i d i gested b r i e f l y  (2 min) w i t h 500 U/ml m i c r o c o c c a l  nuclease,  and was f r a c t i o n a t e d on a 10-30% s u c r o s e g r a d i e n t . sultant  A260  profile  The r e -  ( F i g . 7) shows a main peak ( a t = 1 1 S ) ,  a second peak ( a t = 16S) and a s h o u l d e r o f h e a v i e r m a t e r i a l . C h a r a c t e r i z a t i o n on 2.5% p o l y a c r y l a m i d e g e l s o f t h e DNA e x t r a c t e d from f r a c t i o n s o f t h e g r a d i e n t  (photos i n F i g . 7 ) ,  shows t h a t t h e main 11S peak c o n t a i n s monomer DNA, w h i l e the second peak c o n t a i n s p r e d o m i n a n t l y  dimer DNA, and t h e  s h o u l d e r r e g i o n has even l a r g e r DNA o l i g o m e r s .  Note a g a i n  -96-  8 FRACTIONS  16 FROM  24 TOP, 0.4ml  FIG. 7. C h a r a c t e r i z a t i o n o f DNA i n c h r o m a t i n s u b u n i t s from d i g e s t e d n u c l e i . Chromatin was i s o l a t e d from n u c l e i d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , and f r a c t i o n a t e d on a 5-30% s u c r o s e g r a d i e n t as d e s c r i b e d i n M a t e r i a l s and Methods. M a t e r i a l from the g r a d i e n t f r a c t i o n s was p r e c i p i t a t e d w i t h 10 mM M g , r e s u s pended i n 1% SDS, 20 mM EDTA, 1 M N a C l and e x t r a c t e d w i t h phenol. DNA was p r e c i p i t a t e d w i t h 2 volumes o f e t h a n o l and a n a l y s e d on 2.5% p o l y a c r y a l m i d e g e l s . G e l s were s t a i n e d w i t h " S t a i n s a l l " ; g e l p h o t o s appear above c o r r e s p o n d i n g g r a d i e n t fractions. + +  -97-  t h e 2 bands s m a l l e r t h a n 170-200 base p a i r s l o n g w h i c h on o v e r l o a d e d DNA g e l s  (but i n v e r y l o w y i e l d ,  the u n f r a c t i o n a t e d chromatin (b)  appear  j u d g i n g from  sample).  Sedimentation v e l o c i t y a n a l y s i s of chromatin subunits  As n o t e d i n M a t e r i a l s and Methods, a s o l u t i o n o f c h r o m a t i n s u b u n i t s was sedimented  i n t h e Model E u l t r a e e n t r i f u g e , and  the moving boundary was m o n i t o r e d w i t h t i m e by d i r e c t s c a n n i n g a t 260 nm o r by S c h l i e r e n o p t i c s .  T a b l e 1 shows t h e d i s p l a c e -  ment v s t i m e d a t a f o r (a) 0.8 A2 6o/ml s u b u n i t s by d i r e c t s c a n n i n g , and (b) 60 OD/ml o f s u b u n i t s u s i n g S c h l i e r e n o p t i c s . The s l o p e s o f l o g ( d i s p l a c e m e n t ) v s t i m e p l o t s a r e used t o c a l c u l a t e t h e s v a l u e as f o l l o w s : recall  In r/ro = w s 2  logio  ( t - to)  r / r o = 2^3Q3  S  , where t  ^  ~  t o  ^  i s i n minutes  ' ^ere t i s i n w  seconds So t h e s l o p e o f such a p l o t = 60 w s 2.303 2  AND 2.303 ,., . 1 s = - g Q — ( s l o p e ) -pr  , w = 2* 0  rpm ^ -  .  C o r r e c t i o n s a r e t h e n made f o r t h e d i f f e r e n t d e n s i t y viscosity  (p) and  (n) o f 20 mM T r i s - H C l a t 4°C a s compared t o w a t e r  a t 20°C, [P20,w/p = 1.005,  n/n o,w 2  = 1.52] assuming  V, t h e  p a r t i a l s p e c i f i c volume f o r c h r o m a t i n , i s 0.69 ( 1 4 9 ) . Whereas d i r e c t s c a n n i n g g i v e s i n f o r m a t i o n o n l y on t h e s l o w e s t (- 11S) component, u s i n g S c h l i e r e n o p t i c s , c h r o m a t i n  -98-  TABLE I Chromatin (a)  Scanner  Time  Subunits-Sedimentation Velocity  Optics  (t)  Displacement  0 min  s  2  36,000 rpm; 20°C;  20 mM  (r)  Tris  Logior  5.978 cm  0.7766  18  6.1063  0.7855  32  6.2029  0.7926  45  6.2995  0.7993  62  6.3639  0.8038  78  6.5249  0.8145  0  #  w  = slope x  —  .303/60)  4.38 x 1 0 " " x 2.701 x 1 0 " =  11.8S  9  x  1.005  Data pH  7.6  TABLE (b)  Schlieren Optics  4 0 , 0 0 0 r p m ; 5 ° C ; 20 mM T r i s pH 7 . 6  monomer Time  (t)  I  Displacement  dimer  (r)  Logio r  Displacement  (r)  trimer Logio r  -  -  -  (r)  Logio r  16 m i n  5 . 9 4 4 cm  0.7741  32  6.0217  0.7797  48  6.0993  0.7852  6.193 cm  0.7919  6.292 cm  0.7987  64  6.1792  0.7909  6.320  0.8007  6.452  0.8097  80  6.2616  0.7967  6.438  0.8087  6.607  0.8200  96  6.3486  0.8027  6.556  0.8166  6.767  0.8304  monomer i2  o'  W  = slope *  s  3.4  x  ( 2  - °3/ 3  W  6 0  >  x l O * x 2.187 -  x  nT,b  (l-vp)  T120/W  d-^P)T,b  x 10  _ 9  x 1.52  x  2 0  ,w  1.005  11.3S  dimer S  Displacement  2 0 / W  = 4 . 9 x 10 * x 2.187 x 1 0 "  9  x 1.52  x 1.005  =  16.4S  = 6.6  9  x 1.52  x 1.005  =  22.3S  -1  trimer S o, 2  w  x 10" * x 2 . 1 8 7 1  x 10~  —  -  ID  -100-  16  K  32'  k  64'  48'  FIG. 8. S e d i m e n t a t i o n v e l o c i t y a n a l y s i s o f c h r o m a t i n from d i g e s t e d n u c l e i . Chromatin (60 A2 6 o / m l o f DNA) was i s o l a t e d from n u c l e i d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , and sedimented i n a s i n g l e s e c t o r c e l l a t 36,000 rpm, 5° i n 20 mM T r i s pH 7.6, 0.2 mM EDTA, on a Beckman Model E a n a l y t i c a l U l t r a c e n t r i f u g e u s i n g S c h l i e r e n o p t i c s . Photos were t a k e n a t 16 minute i n t e r v a l s d u r i n g the r u n , and S o v a l u e s were c a l c u l a t e d as d e s c r i b e d i n M a t e r i a l s and Methods. The phase p l a t e a n g l e was 50°. 2  w  -101-  from d i g e s t e d n u c l e i s e p a r a t e d  ( F i g . 8) i n t o s e v e r a l s p e c i e s ;  a major peak a t l i s (monomer), a second peak a t 16S (dimer) and a t h i r d a t 22S ( t r i m e r ) .  The symmetry o f t h e peaks  argues s t r o n g l y f o r homogeneity i n s i z e , i f n o t c o m p o s i t i o n . These s i z e e s t i m a t e s c o r r e l a t e w e l l w i t h t h o s e o b t a i n e d on s u c r o s e g r a d i e n t s ; however, t h e i o n c o n c e n t r a t i o n p r e s e n t (20 mM T r i s - H C l ) may have been i n s u f f i c i e n t t o p r e v e n t anom a l i e s due t o s a l t (c)  effects.  Sepharose 2B chromatography  Chromatin  s u b u n i t s i n 0.2 mM EDTA c o u l d a l s o be r o u g h l y  f r a c t i o n a t e d on a Sepharose 2B column ( F i g . 9 ) , a g a i n i n low s a l t , 5 mM T r i s - H C l pH 7.6, 0.2 mM EDTA.  Monomers e l u t e d i n  a p o s i t i o n corresponding t o a molecular weight o f a p p r o x i m a t e l y 400,000.  This i s s l i g h t l y higher than expected f o r  an 11S p a r t i c l e c o n t a i n i n g 200 base p a i r s o f DNA and an approximately equal weight o f h i s t o n e s .  T h i s may i n d i c a t e  d e v i a t i o n o f t h e s u b u n i t s from a s p h e r i c a l shape, o r a l a r g e h y d r a t i o n s h e l l f o r t h e presumably h i g h l y charged s u b u n i t s . :i.  T r o u t T e s t i s Chromatin N u c l e o h i s t o n e and  Subunit S t r u c t u r e :  Comparison o f  Nucleoprotamine  T r o u t t e s t i s d i f f e r s from o t h e r t i s s u e s s t u d i e d s i n c e i t undergoes a change from m i t o t i c t o m e i o t i c t i s s u e w i t h subsequent replacement was  o f n u c l e o h i s t o n e by n u c l e o p r o t a m i n e .  It  o f i n t e r e s t t h e r e f o r e t o see how t h e s e changes would be  r e f l e c t e d i n t h e DNA p r o d u c t s o f m i c r o c o c c a l n u c l e a s e  digestion.  -102-  FRACTION  NO.,  2.0 ml  FIG. 9. Sepharose 2B chromatography o f chromatin i s o l a t e d from nuclease-digested n u c l e i . Chromatin (10-100 A2 6 o / m l o f DNA) from n u c l e a s e - d i g e s t e d n u c l e i was loaded on a 2.5 x 40 cm Sepharose 2B column e q u i l i b r a t e d and run i n 0.2 mM EDTA, 5 mM T r i s pH 7.6. F r a c t i o n s o f 2.0 ml were c o l l e c t e d and A 2 6 0 was monitored. The approximate e l u t i o n p o s i t i o n s o f E. c o l i B - g a l a c t o s i d a s e (MW = 540,000) and bovine c a t a l a s e (MW = 240,000) are marked by arrows.  -103-  When sperm n u c l e i , which c o n t a i n o n l y nucleoprotamine, are d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , no DNA fragments a r e produced  ( F i g . I c ) , w i t h u n d i g e s t e d DNA remaining a t the g e l  origin.  No 11S chromatin s u b u n i t s c o u l d be prepared from  sperm n u c l e i . We next examined t r o u t t e s t i s a t i n t e r m e d i a t e stages o f development,  c o n t a i n i n g both n u c l e o h i s t o n e and nucleoprotamine.  In e a r l y protamine  stage t e s t i s , much o f the t i s s u e i s  m e i o t i c i n o r i g i n and spermatid c e l l s , which have j u s t making protamine, predominate  (93).  from such t i s s u e i s comparable mitotic testis,  The y i e l d o f DNA  fragments  t o y i e l d s from h i s t o n e stage  s u g g e s t i n g t h a t m e i o t i c and m i t o t i c  have a s i m i l a r h i g h p r o p o r t i o n o f s u b u n i t s t r u c t u r e When mid-protamine  started  chromatin (Table I I ) .  stage n u c l e i a r e d i g e s t e d w i t h m i c r o -  c o c c a l n u c l e a s e , DNA fragments a r e produced  i n an amount  r o u g h l y p r o p o r t i o n a l t o the amount o f n u c l e o h i s t o n e i n i t i a l l y present  (Table I I ) . The l i s  s u b u n i t s from such t i s s u e con-  t a i n e d o n l y h i s t o n e s , w i t h n e g l i g i b l e protamine.  These  results  i n d i c a t e t h a t t h e s t r u c t u r e s o f nucleoprotamine and n u c l e o h i s t o n e must be v e r y d i f f e r e n t . :v.  P r o t e i n Composition o f Chromatin  Subunits  Having looked a t t h e s i z e and DNA c o n t e n t o f chromatin s u b u n i t s , we next examined the p r o t e i n complement o f these subunits.  S u b u n i t s showed a protein:DNA  mately 1.2 and an  A 2 3 o : A  2  5 8  r a t i o of approxi-  r a t i o o f 0.72, both lower than  -104-  TABLE I I  Developmental stage  %  Histone early mid late  The from  100  protamine  protamine protamine  mature  Nucleohistone  sperm  % fragmented  DNA  % Nucleoprotamine  -•  80  95  5  80  35  65  28  5  95  2  -  100'  1  from d i g e s t e d  n u c l e i was  2.5% p o l y a c r y l a m i d e g e l s o f e x t r a c t e d  atively,  chromatin  from  determined  DNA.  s u b u n i t s were p r e p a r e d a n d DNA  by t h e d i p h e n y l a m i n e quantitated  % DNA released into fragments  reaction.  Alterndetermined  H i s t o n e s and p r o t a m i n e  15% u r e a - p o l y a c r y l a m i d e g e l s .  were  -105-  values  (1.5 and  0.8  r e s p e c t i v e l y ) r e p o r t e d f o r nuclease d i -  g e s t s o f i s o l a t e d chromatin The  (149).  f i v e major h i s t o n e s p e c i e s , as w e l l as the t r o u t -  s p e c i f i c h i s t o n e H6,  c o u l d be i d e n t i f i e d on g e l s from  f r a c t i o n a t e d or f r a c t i o n a t e d s u b u n i t s . chromatin s u b u n i t s were p r e s e n t  These h i s t o n e s  i n roughly  t i o n s as i n whole chromatin i . e . roughly by p o l y a c r y l a m i d e  Across  (157)  a sucrose  and  un-  starch gel  the same  propor-  equimolar, as  (120)  of  judged  electrophoresis.  g r a d i e n t f r a c t i o n a t i o n o f chromatin  s u b u n i t s , however, the monomer peak was  relatively  deficient  i n h i s t o n e I ( F i g . 10), w i t h an excess of f r e e h i s t o n e I appearing  a t the top o f the g r a d i e n t .  h i s t o n e r a t i o o f 0.2  obtained  The  histone  I/total  from Coomassie Blue s t a i n i n g  on g e l s i s somewhat h i g h , s i n c e the l a r g e r h i s t o n e I b i n d s p r o p o r t i o n a t e l y more N o l l had  dye.  reported  (147)  the presence o f non-histone  p r o t e i n s on chromatin subunits  from r a t l i v e r n u c l e i .  Such  p r o t e i n s have been r e p o r t e d i n amounts up to equal weight w i t h the h i s t o n e s and  DNA.  As t h e r e i s g r e a t i n t e r e s t i n these  p r o t e i n s as p o s s i b l e gene r e g u l a t o r s , we  looked  h i s t o n e p r o t e i n s i n t e s t i s chromatin s u b u n i t s . histone proteins  (> 25,000 m o l e c u l a r  by s t a i n i n g techniques  f o r nonNo  non-  weight) were d e t e c t a b l e  on SDS-polyacrylamide g e l s , even when  -106-  0  8  FRACTIONS  16  FROM  24  TOP,  0.4ml  F I G . 10. H i s t o n e c o n t e n t i n c h r o m a t i n from d i g e s t e d n u c l e i , f r a c t i o n a t e d on a s u c r o s e g r a d i e n t . C h r o m a t i n was i s o l a t e d f r o m n u c l e i d i g e s t e d w i t h m i c r o c o c c a l n u c l e a s e , a n d was f r a c t i o n a t e d o n a 5-30% s u c r o s e g r a d i e n t . C h r o m a t i n was m o n i t o r e d by i t s a b s o r b a n c e a t 260 nm. P r o t e i n s were e x t r a c t e d i n 2% SDS f r o m M g p r e c i p i t a t e s o f g r a d i e n t f r a c t i o n s and e l e c t r o p h o r e s e d on SDS 5% p o l y a c r y l a m i d e g e l s . G e l s were s t a i n e d w i t h C o o m a s s i e B l u e and s c a n n e d a t 550 nm on a G i l f o r d spectrophotometer. H i s t o n e I and t o t a l h i s t o n e were q u a n t i t a t e d from scan a r e a s . + +  -107-  s u b u n i t s were e x t r a c t e d w i t h 5% SDS loaded f o r h i s t o n e s .  and g e l s h e a v i l y o v e r -  To e x p l a i n t h i s  e i t h e r - ( i ) o t h e r r e g i o n s o f DNA,  result,  w h i c h a r e n o t c o v e r e d by  h i s t o n e s i n a subunit s t r u c t u r e , c o n t a i n a l l the non-histone p r o t e i n s , o r ( i i ) t r o u t t e s t i s does n o t c o n t a i n n o n - h i s t o n e p r o t e i n s i n the q u a n t i t i e s reported f o r other t i s s u e s . To d i s t i n g u i s h t h e two p o s s i b i l i t i e s , n u c l e i o r i s o l a t e d c h r o m a t i n were p r e p a r e d and t h e n o n - h i s t o n e p r o t e i n s were e x t r a c t e d w i t h 1% SDS  from c h r o m a t i n f o l l o w i n g HC1  and o r g a n i c  s o l v e n t t r e a t m e n t (162) . Such n o n - h i s t o n e p r o t e i n s make up l e s s t h a n 5 % o f t h e h i s t o n e s by w e i g h t i n t h i s t i s s u e , even i n t h o s e e a r l y s t a g e s where t h e s t r u c t u r e o f c h r o m a t i n and c e l l u l a r (DNA  events  & histone synthesis, mitosis) are otherwise s i m i l a r to  those of o t h e r organisms. V.  The B e h a v i o u r o f C h r o m a t i n S u b u n i t s i n S a l t I t was n o t i c e d f a i r l y e a r l y t h a t s u b u n i t s , l i k e  isolated  c h r o m a t i n , c o u l d be caused t o p r e c i p i t a t e by a d d i t i o n o f Mg (10 mM). analysis.  T h i s was used t o c o n c e n t r a t e samples f o r b i o c h e m i c a l F i g u r e 11 shows t h e p r e c i p i t a t i o n o f s u b u n i t s as  a s s a y e d by t u r b i d i t y  (Ai*4o)  t r i f u g a t i o n a t 1,500  g, 10 min)  T r i s ) and  o r decrease i n  A260  f o r (a) monovalent (Na , K ,  (b) d i v a l e n t ( M g , C a ) c a t i o n s . 2 +  ( a f t e r cen-  2 +  -108-  *  0  *  2  •  -  6  CaCI , rnM  '  10  2  FIG. 11. P r e c i p i t a t i o n of c h r o m a t i n s u b u n i t s by monovalent and d i v a l e n t c a t i o n s . C h r o m a t i n s u b u n i t s (1.5 A2 6o/ml) were t r e a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f (a) N a C l , T r i s - H C l o r (b) C a C l 2 , M g C l and t u r b i d i t y a t 440 nm was m o n i t o r e d . Then samples were c e n t r i f u g e d a t 4000 rpm, 10 m i n u t e s and s u p e r n a t a n t absorbance a t 260 nm was measured. 2  -109-  Monovalent tation  of subunits  divalent units than  cations  cations  n e a r 1 mM Mg  + +  (Mg  , Ca  (Fig. 5), C a  Tris)  T  effected  ) precipitated + +  being  50% p r e c i p i -  7 0 mM,  while  50% o f t h e s u b -  s l i g h t l y more  effective  .  Mg*  that across  a sucrose  p e l l e t s o f t h e monomer f r a c t i o n s  tions  8-10) were t h i n  tions  gave  and c l e a r ,  opaque w h i t e p e l l e t s .  o f monomer p e l l e t s of  -1  a t c o n c e n t r a t i o n s around  I t was n o t e w o r t h y units,  (Na ", K ,  fractions.  ( F i g . 11, f r a c -  whereas t h e o l i g o m e r The d i f f e r e n t  correlates well with  histone I i n these  g r a d i e n t o f sub-  frac-  appearance  the r e l a t i v e  absence  -110-  DISCUSSION T h i s t h e s i s supports  the c u r r e n t p i c t u r e o f chromatin  "beads on a s t r i n g " , i . e . segments of DNA c l u s t e r s o f h i s t o n e s and DNA  separated  as  associated with  by exposed spacer  DNA.  Digestion Patterns Our  r e s u l t s show t h a t m i c r o c o c c a l nuclease  d i g e s t i o n of  i n t a c t t r o u t t e s t i s n u c l e i g i v e s r i s e to approximately base p a i r long fragments o f DNA (=400, 600  result  (monomer) and m u l t i p l e s t h e r e o f  e t c . ) , as had been noted by Hewish and  (146), N o i l  (147)  and L o u i e  (148).  Burgoyne  The u n i v e r s a l i t y o f  [ d e s c r i b e d i n r a t (146), mouse (148), polyoma  b i r d erythrocyte  200  (166), c a l f thymus  (7), y e a s t  this  (148),  (7), t r o u t  t e s t i s and o t h e r organisms*] i s becoming c l e a r . One DNA  would not expect  s i z e , g i v e n the s i m i l a r i t y i n h i s t o n e composition  chromatin  Holde and coworkers  (7) have r e p o r t e d t h a t monomer  i s 135-150 base p a i r s l o n g .  "trimming" o f DNA dimer and by N o l l  and  p r o p e r t i e s a c r o s s the p h y l o g e n e t i c t r e e .  Van DNA  g r e a t d i f f e r e n c e s i n t h i s monomer  T h i s may  be due  t o more  from the monomer s i n c e f o r c a l f thymus the  t r i m e r bands c l o s e l y approximate the s i z e s r e p o r t e d  (147).  the s i z e s of DNA  There may  a l s o be d i f f e r e n c e s i n c a l i b r a t i o n  fragments between the two  r e s e a r c h groups.  * Reported a t the EMBO Symposium on Developmental G e n e t i c s , H e i d e l b e r g , May 1975  of  -Ill-  All  these r e s u l t s point  ment o f p r o t e i n s by  t o an o r d e r e d  repetitive  o n 170 b a s e p a i r s o f DNA,  nuclease-sensitive  DNA  "spacer"  i n units  regions  arrangeseparated  o f about  35 b a s e  pairs. We long  obtain  DNA  ( F i g . 6) o n l y  nuclei. within,  as opposed  isolated  than about  low y i e l d  reflect  (up t o one h o u r ) m i c r o c o c c a l chromatin,  reported  fragments.  p r o d u c t s by A x e l  digestion of  45-130 b a s e  b y 10-15 b a s e p a i r i n t e r v a l s . internal  cleavage within  Such  l o n g e r ) , ending with  50-150 b a s e p a i r s l o n g . through  that  intimate  digests long  DNA  (154) h a s shown t h a t  into discrete pieces  a t 10 n u c l e o t i d e  intervals.  from  proceeds  t o a t r i m m e d monomer of 8 limit  f r a g m e n t s may  contact  (166) a n d  such d i g e s t i o n  the production These  pairs  fragments  with  DNA  represent  the histones  167), p o s s i b l y v i a t h e N H 2 - t e r m i n i o f t h e s e p r o t e i n s N o l l ' s work  t o 100  a chromatin sub-  Indeed, Sollner-Webb, F e l s e n f e l d  (167) h a v e r e c e n t l y r e p o r t e d  o f DNA  using  e t a l . (155) a n d W e i n t r a u b a n d V a n L e n t e  t h r o u g h a 185 b a s e p a i r monomer DNA,  tion  (149),  and  Refinement o f t h e a n a l y s i s o f d i g e s -  separated  u n i t monomer.  sites  Clark  t h a t t h e DNA was d i g e s t e d  long,  and  at additional  nuclease  showed them t o be d i s c r e t e f r a g m e n t s  presumably r e f l e c t  pairs  digestion of  subunits.  (156)  Axel  after  cleavage  t o between c h r o m a t i n  170 b a s e  (165) a n d S a h a s r a b u d d h e a n d V a n H o l d e  base p a i r long tion  smaller  i n extremely  These a p p a r e n t l y  Felsenfeld extended  fragments  another  (140  fragments, protec(166,  (156).  enzyme, DNase I ,  10 t o 200  nucleotides  T h i s work s t r o n g l y  suggests  -112-  that  t h e DNA  i n a chromatin  of a histone the  core,  subunit.  with  As p o i n t e d  i s covered  from a p a r t i c l e  —'  10  10 •  1  to give  coccal nuclease  testis  products: and  1  t  .  cleavage  points  chromatin  1  and t h e i r  shearing size (170)  (200 b a s e p a i r s ) ,  have s i n c e r e p o r t e d DNA c a n be  Micrococcal rise  been  multiples, but the relation  to micro-  Noll  nuclei)  nuclease  from  digestion  t r i m m e d monomer  (170)  e t a l . (169) h a v e r e p o r t e d  o f chromatin  from  that  from  that  chromatin;  included  F e l s e n f e l d and c o w o r k e r s  Protein Composition  gives  t  extensive  p r o c e d u r e s w h i c h w o u l d c a u s e a s m e a r i n g among  classes.  monomer  f  base p a i r length  i s unclear..  t h e same m i c r o c o c c a l  preparation  10  1  i  (as opposed t o i n t a c t  (Fig. 5).  10  1  DNase I I (168) h a s a l s o  such d i s c r e t e fragments a r e n o t recovered however t h e i r  10 1  l  DNA a n d h i g h e r  smaller  fragments  gives  10  1  a monomer  monomer  multiples  i n w h i c h much o f t h e  40  1  fragments a r e apparently  trout  fragments  s  40  Digestion of chromatin with  Isolated  within  covered  d  MM,  reported  feature  by h i s t o n e s : - e . g .  / 10 '  structural  the outside  o u t b y C r i c k ^ however, s u c h  essentially 10  i s wound a r o u n d  some r e p e t i t i v e  m i g h t a l s o be g e n e r a t e d DNA  subunit  DNA  (166,167), O u d e t e t a l .  isolated  chromatin d i s c r e t e  prepared.  and Arrangement  nuclease  i n Chromatin Subunit  digestion of intact  t o 11S c h r o m a t i n p a r t i c l e s  ^ F.H.C. C r i c k , p e r s o n a l  communication  testis  Particles  nuclei  (monomers) c o n t a i n i n g  -113-  a p p r o x i m a t e l y 200 base p a i r l o n g fragments o f DNA and a s s o c iated histones.  P r e s u m p t i v e c h r o m a t i n d i m e r s (16S) and t r i m e r s  (22S) r e s u l t i n g from i n c o m p l e t e d i g e s t i o n c o u l d a l s o be i s o l a t e d , c o n t a i n i n g c o r r e s p o n d i n g l y l o n g e r DNA p i e c e s ( F i g . 7,8). E v i d e n c e i s c o n v e r g i n g now t h a t such p a r t i c l e s , r e p o r t e d by Van Holde and coworkers  (149) and N o l l  first  (147) a r e  indeed r e p r e s e n t a t i v e of chromatin s t r u c t u r e i n the nucleus. Bradbury and coworkers  (171) have d e m o n s t r a t e d , by n e u t r o n  d i f f r a c t i o n s t u d i e s , t h a t t h e r e f l e c t i o n s a t 110, 55, 37, 27 o  A o b s e r v e d i n X-ray s t u d i e s c a n be b e t t e r e x p l a i n e d , n o t as a s u p e r c o i l , b u t as a g l o b u l a r mass o f p r o t e i n  (accounting f o r  o  the 110, 37 A r e f l e c t i o n s ) a s s o c i a t e d w i t h DNA.  Electron  m i c r o s c o p y o f SV40 v i r u s m i n i chromosomes ( 1 7 2 ) , and c h r o m a t i n o  from v a r i o u s s o u r c e s (150,170,173) c l e a r l y show =100 A beads o f D N A - p r o t e i n a l o n g a s t r i n g o f DNA.  Such r e s u l t s  o  t h a t =200 base p a i r s  ( = 680 A) a r e compacted  indicate o  i n t o =100 A  f o r a p a c k i n g r a t i o o f a p p r o x i m a t e l y 7:1. How do t h e p r o t e i n s and DNA i n t e r a c t t o produce such a packing r a t i o ? t h e DNA  F o r t r o u t t e s t i s c h r o m a t i n , from t h e s i z e o f  (200 base p a i r s =130,000 d a l t o n s ) , t h e S v a l u e (11S),  and t h e protein:DNA r a t i o  (1.2:1), e i g h t t o nine h i s t o n e  m o l e c u l e s would be e x p e c t e d p e r c h r o m a t i n monomer.  Our r e s u l t s  demonstrate t h a t s u b u n i t monomers ( F i g . 11) a r e d e f i c i e n t i n histone H i .  This r e s u l t correlates w e l l with the observations  t h a t removal o f HI r e s u l t s i n n e g l i g i b l e changes i n c h r o m a t i n s t r u c t u r e , when a n a l y z e d by p h y s i c a l means (66-68).  Indeed,  -114-  nuclease the  d i g e s t i o n s performed  same r e s u l t s  t o be for  roughly  (148,170).  equimolar  the presence  histone  I)  o f two  as was  on H l - d e f i c i e n t The  fact  i n chromatin  chromatin  t h a t the h i s t o n e s s u b u n i t s would  by  Kornberg  (152).  appear  argue  o f each o f the major h i s t o n e s  suggested  give  (except  Heterogeneity  w i t h r e s p e c t t o w h i c h s p e c i e s o f h i s t o n e a r e p r e s e n t on i n dividual  chromatin  crosslinking crude  subunits i s not  r e s u l t s w o u l d be  t o p o g r a p h i c a l map  done w i t h  ribosomes  of  subunits proceeds  2 e a c h o f H2A,  H2B,  up  H.3 and  t o an H4.  These h i s t o n e o l i g o m e r s  chromatin an  chromatin  are  2  2  tetramer  i a t e d w i t h DNA.  The  lead  associated with Lente  gests that histones' basic N-termini the non-polar  has  subunits a mixture  and  to those  prepared  to a p i c t u r e of  of  8 histones  a  (perhaps  two  H2A-H2B d i m e r s )  assoc-  and  Weintraub  sug-  (156)  DNA  such  an  sequence s t u d i e s  undergo  b e e n d e s c r i b e d f o r r i b o s o m e s and of  consist  while  regions allow histone-histone i n t e r a c t i o n ,  Such a s u b u n i t m i g h t a l s o  support  (174)  isolated  interact with  a c c o r d w i t h t h e p r e d i c t i o n s made f r o m 23).  been  observed.  similar  work o f Van  Kornberg  o c t a m e r , w h i c h may  s u b u n i t as a g l o b u l a r c l u s t e r  (H3) (H4)  in obtaining a  H2A-H2B d i m e r s  (32-34) and  Protein  s u b u n i t , as has  of the h i s t o n e s of  2  isolated  however.  Thomas and  ( H 3 ) ( H 4 ) 2 ( H 2 A ) ( H 2 B ) hexamers were a l s o  from  out  useful  chromatin  Recently  have shown t h a t c r o s s l i n k i n g chromatin  extremely  of the  (22).  ruled  "self-assembly"  certain  viruses.  idea, recent reconstitution  (to g i v e the  same n u c l e a s e  o f h i s t o n e s and  DNA  has  of  in  (15,17, as In  chromatin  d i g e s t i o n p a t t e r n s ) , from  been r e p o r t e d  (155,170).  -115-  The  l o c a t i o n and t y p e o f f o l d i n g o f DNA i n a s u b u n i t a r e  s t i l l unclear.  N o l l ' s r e s u l t s w i t h DNase I (154) appear t o  show t h a t DNA i s on t h e o u t s i d e o f a s u b u n i t .  Such a p i c t u r e  w o u l d a l s o a l l o w f o r i n t e r a c t i o n s o f DNA w i t h o t h e r m o l e c u l e s (enzymes e t c . ) .  C r i c k and K l u g have proposed an a t t r a c t i v e  model f o r t h e p a c k i n g o f DNA i n t o a s u b u n i t  (175).  The DNA  i n t h i s model c a n be " k i n k e d " a t about 100°, r e s u l t i n g i n a l e f t - h a n d e d h e l i x o f 20 base p a i r l o n g s t r a i g h t s t r e t c h e s sepa r a t e d by k i n k s . The n a t u r e o f s p a c e r DNA between s u b u n i t s i s a l s o somewhat "fuzzy".  The amount o f DNA i n a s u b u n i t i s g e n e r a l l y  t o be a p p r o x i m a t e l y  accepted  200 base p a i r s l o n g , i n c l u d i n g a s p a c e r  r e g i o n o f about 30-40 n u c l e o t i d e s .  However, t h e r e have been  r e p o r t s (7,166,167) t h a t t h e l i m i t d i g e s t o f monomer has a DNA length^" o f a p p r o x i m a t e l y  140-150 base p a i r s ( i . e . 50-60  base p a i r s have been l o s t ) .  I t may be t h e n t h a t a f t e r  DNA c l e a v a g e trimming  initial  t o produce a 200 base p a i r l o n g f r a g m e n t , a s l o w e r  o f spacer  (30-40) o c c u r s , f o l l o w e d by d i g e s t i o n o f  even l e s s a c c e s s i b l e DNA t o 140-150 base p a i r s . E l e c t r o n micrographs o f chromatin  show t h a t t h e s p a c e r  DNA r e g i o n s a r e e x t e n d e d , and v i s i b l e as " t h r e a d s "  between 2  s u b u n i t "beads" (150,170,173).  F i n c h and c o w o r k e r s  at  F.H.C. C r i c k , p e r s o n a l communication R e p o r t e d a t EMBO Symposium on Developmental G e n e t i c s , H e i d e l b e r g , 1975  -116-  C a m b r i d g e , however, h a v e c l a i m e d in  c o n t a c t with each other;  of  sample p r e p a r a t i o n . Histone  H i , as noted  that subunits are normally  linear  earlier,  spacer  DNA  probably  i s an  i s not necessary  for maintaining the basic subunit structure of The  absence o f H i from  i n higher oligomers  testis  h a v e some c r o s s - l i n k i n g  chromatin.  s u b u n i t monomers, a n d  ( F i g . 11) s u g g e s t s  artifact  presence  t h a t H i may i n s t e a d  f u n c t i o n (69-71).  This histone could  c o n t a c t p r o t e i n s b e t w e e n i n d i v i d u a l monomers o r r e s i d e i n t h e DNA s p a c e r  regions, maintaining chromatin  i n a condensed  I n d e e d , Chambon a n d c o w o r k e r s have r e p o r t e d e l e c t r o n micrographs closely  in  (170) t h a t i n  subunits with Hi present are  packed.  The reason  o f chromatin,  crosslinking  of free  f o r the presence  the l a r g e r chromatin  s u b u n i t s b y h i s t o n e I may be t h e  o f s m a l l amounts o f monomer DNA oligomers  (Fig. 7 ) , although  reflect  aggregation during gradient centrifugation.  histone  interactions  alone  t h e m a j o r i t y o f monomer  Noll  (147), The  pellets  from  (e.g.  allowing may p r o v e  these  an o r d e r e d useful  coincides with  preparations.  DNA w i t h  may  Histone-  i n t h e 11S monomer  absence o f h i s t o n e I i n chromatin  as opposed t o o l i g o m e r s ,  this  subunits together, as noted  a s m o s t monomer DNA r u n s  relative  even  are evidently not s u f f i c i e n t t o  hold  metry  form.  the thin  region.  monomers,  clear  T h i s may r e f l e c t  by  Mg  some sym-  eight histones i n a regular structure)  packing  i n physical  o f monomers.  T h e s e monomer  studies of subunit  pellets  structure.  -117-  One  possible  problem  might  be  the presence  monomer p o p u l a t i o n s , v a r y i n g i n s i z e 1  (=200 b a s e p a i r s )  pairs)  to  d i g e s t i o n ) might  (147)  had  were p r e s e n t i n 11S However, we trout  testis  chromatin  chromatin  and  Rusch  SDS  extraction.  served  reported that  could find  (161), E l g i n A few  no  and  proteins observed equal weight  by  functions may  rather  contaminants  This  testis  f o r the  seems u n l i k e l y  ongoing  proteins  —  proteins  processes  in  LeStourgeon direct  hot  5%  s p e c i e s c o u l d be  then, that  e.g.  protein  as  approaching  nuclear proteins (137),  the  or  special  "shutting  down" g e n e t i c  protamine  into  sperm  the  —  proteins.  stages of  s y n t h e s i s and  (93) , and  ob-  non-histone  f r o m membranes  Alternatively,  and  the  levels  ( 1 3 5 ) , c o u l d be  however f o r t h e e a r l y RNA,  nuclei.  t h a n c h r o m a t i n , were u s e d  a tissue o f DNA  (NHP)  rat liver  lack of observable non-histone  d e v e l o p m e n t , when DNA, are s t i l l  (138).  in a  .  ( 1 6 2 ) , o r by  very minor p r o t e i n  of  result  t h e methods o f  some w o r k e r s a t t o t a l  f o r packaging  account  + +  non-histone  Bonner  with the h i s t o n e s  nature of trout  Mg  s u b u n i t s from  not associated with chromatin, cytoplasmic  of  I t seems l i k e l y by  limited  presence  detectable non-histone  either  i f washed n u c l e i ,  starting material.  The  c o n f e r asymmetry and  complex i n the p r e s e n c e  Noll  (such as  base  g i v e a more homogeneous p o p u l a t i o n  other studies.  I i n o l i g o m e r s may  disordered  t h o s e c o n t a i n i n g band  Further treatment  o f monomer f o r x - r a y and histone  two  t h o s e c o n t a i n i n g band l a (=170  o r s m a l l e r DNA.  exonuclease  from  of at least  testis  cell  structure  and  division func-  -118-  tion  of chromatin  are  similar  to other  tissues.  Instead,  l a r g e n u c l e a r / c y t o p l a s m i c r a t i o makes i t r e l a t i v e l y prepare  chromatin  the  levels  low  a c t i n , myosin  f r e e of contaminants;  o f NHP (176)  present.  and  present  t h a t any  i n the  early  chromatin  has  Testis  pre-meiotic the  stages  same s u b u n i t  other organisms.  Our  results  histone of meiotic tissue  has  portion  cycle.  of c e l l s  I t may  be  undergoing  of t e s t i s  indicate a similar  need not  be  development, i t s that reported  in different  f o r example t h a t t h e  nucleo-  served  a r r e s t e d i n metaphase; the  stages  small  d i v i s i o n w o u l d show  pro-  differ-  Such d i f f e r e n c e s have been  give different,  for  subunit structure.  structure.  chromosomes a p p a r e n t l y  the  f u r t h e r t h a t the  ences i n s u b u n i t for cells  present;  some w o r k e r s .  s t r u c t u r e as  cell  like  Maturation  Such r e s u l t s a r e an average a c r o s s c e l l s of the c e l l  for  some p r o t e i n s  i . e . NHP,  l a r g e amounts r e p o r t e d by  to  account  proteins involved i n  r e g u l a t i o n o f gene e x p r e s s i o n  Chromatin S t r u c t u r e During At  Certainly  may  n u c l e a r enzymes must be  however, i t seems l i k e l y specific  this  easy  the  ob-  h i g h l y condensed  larger  DNA  fragments  upon m i c r o c o c c a l n u c l e a s e d i g e s t i o n ^ . N u c l e o p r o t a m i n e was analogous to those simply both  an  inhibitor  h i s t o n e and  not  d i g e s t e d t o p r o d u c e DNA  for nucleohistone. of the nuclease  protamine, subunits  F.H.C. C r i c k , p e r s o n a l  P r o t a m i n e was  because i n t i s s u e c o u l d be  communication  fragments not containing  recovered  in  -119-  amounts p r o p o r t i o n a l II).  t o t h e amount o f h i s t o n e p r e s e n t  Nucleoprotamine  analogous  may  to n u c l e o h i s t o n e , but  covered with protamine, is  p r e s e n t , s p a c e r DNA  may  be  n o t t h e n have a s u b u n i t  sterically  or  instead  either  inaccessible.  condensed  structure  ( i ) a l l DNA  ( i i ) i f a subunit-like  in tightly  (Table  is  structure  nucleoprotamine  This t i g h t l y  condensed  struc-  t u r e o f n u c l e o p r o t a m i n e has been n o t e d  from o t h e r  F o r e x a m p l e , when c h r o m a t i n  b o t h n u c l e o h i s t o n e and  nucleoprotamine into  i s sheared, only nucleohistone i s s o l u b i l i z e d  s m a l l e r fragments It  containing  (177).  i s e v i d e n t then t h a t  t h e p a c k i n g o f DNA sperm head.  The  t h e r e i s something  w i t h protamine  which  DNase I I .  One  do n o t c o n t a i n p r o t a m i n e .  interesting  sperm c e l l s I t remains  seen whether o t h e r mechanisms o f c o n d e n s a t i o n i n such cells  will  render the chromatin  about  nucleoprotamine might  the s t r u c t u r e of nucleohistone i n those  goldfish)  unique  i n t o t h e s m a l l volume o f  structure of this  seen v i a o t h e r n u c l e a s e s e.g. is  studies.  inaccessible  a  be problem  (e.g. to  be  sperm  to micrococcal  nuclease.  Conclusion The nuclei which  availability  from t r o u t strongly  chromatin. plan  further  We  o f l a r g e amounts o f r e a d i l y  testis  has  support the now  greatly  "bead  facilitated  on a s t r i n g "  prepared these  picture  have a c o n c e p t u a l framework around  studies  on:  studies, of which  to  -120-  (i)  the nature of the DNA  t h a t DNA  (the o t h e r 15%)  (85%)  complexed w i t h p r o t e i n , and  which i s d i g e s t e d by nuclease i . e .  i n which k i n d of DNA.are the " a c t i v e " genes?  One  approach to  t h i s q u e s t i o n i s v i a h y b r i d i z a t i o n o f s p e c i f i c RNA to  subunit  regions.  ( h i s t o n e - p r o t e c t e d ) DNA A x e l e t a_l.  genes are p a r t i a l l y  (178)  (80%)  or to DNA  ribosomal  containing  spacer  have r e c e n t l y r e p o r t e d t h a t g l o b i n  covered  by p r o t e i n s .  t h a t the presence o f p r o t e i n on DNA strict transcription.  transcripts  They  conclude  i s not s u f f i c i e n t t o r e -  S i m i l a r r e s u l t s have been reported"'' f o r  rRNA genes, although  i n t h i s case the r e s u l t s may  be  e q u i v o c a l , because m u l t i p l e c o p i e s o f the rRNA genes e x i s t i . e . 99 of 100  c o p i e s may  w h i l e the 1 per 100 (ii) new  be p r o t e i n - c o v e r e d  sequences covered  g e n e t i c i n f o r m a t i o n i s expressed. by p r o t e i n ?  r e c o g n i z e d and  expressed),  c o u l d be open and t r a n s c r i b e d ;  the changes i n DNA  covered  (and not  Are  by h i s t o n e s , when specific  sequences  I f genes are p r o t e i n - c o v e r e d , how  activated?  For SV40 v i r a l DNA  are  t h e r e appears t o  be no sequence s p e c i f i c i t y f o r h i s t o n e b i n d i n g , based on random cleavage enzymes (iii)  o f SV40 "minichromosomes" by  the  restriction  (179); the presence o f non-histone  r o l e i n gene e x p r e s s i o n . sence o f NHP  proteins  (NHP)  and  their  I f bur r e s u l t s on the r e l a t i v e  prove u n i v e r s a l , then r a d i o a c t i v e - l a b e l l i n g  [ S]methionine, 35  they  1  2  5  I , P) 32  may  be necessary  Chambon, P. e t a l . , and Jones, A. and results  to d e t e c t  Reeves, R.,  ab(with  these  unpublished  -121-  p r o t e i n s and t h e i r d i s t r i b u t i o n  i n s u b u n i t monomers and h i g h e r  oligomers; (iv)  the l o c a t i o n and mode o f a c t i o n o f the enzymes r e s p o n s i b l e  f o r gene e x p r e s s i o n enzymes, e t c . ) .  (DNA & RNA polymerase, n u c l e a s e s  modifying  F o r example, does m i c r o c o c c a l nuclease d i -  g e s t i o n o f spacer  DNA r e l e a s e enzymes  (RNA polymerase?)  assoc-  i a t e d v/ith t h a t spacer DNA? (v)  t h e assembly o f chromatin  subunits.  I t may be t h a t t h e  h i s t o n e s and DNA w i l l assemble i n v i t r o i n an o r d e r e d r e a c t i o n s , much as t h e ribosome does f i e d o r unmodified portance  (180).  series of  The use o f modi-  h i s t o n e s may show something about t h e im-  o f a c e t y l a t i o n , p h o s p h o r y l a t i o n e t c . i n assembly.  Such i n v i t r o r e c o n s t i t u t i o n experiments may a l s o show how h i s t o n e H i c o n t r i b u t e s t o t h e o r g a n i z a t i o n and p a c k i n g o f subu n i t s i n t o a higher order s t r u c t u r e . I t would a l s o be o f i n t e r e s t t o see, v i a [ *C] amino a c i d 11  and  [ H ] t h y m i d i n e l a b e l l i n g , t h e time course 3  incorporation into subunits. c r e a s i n g l a b e l l i n g time,  of r a d i o a c t i v e  One might p r e d i c t t h a t w i t h i n -  [ H ] t h y m i d i n e i n a c i d - s o l u b l e (spacer) 3  DNA should r i s e and p l a t e a u , w h i l e H i n s u b u n i t 3  (protected)  DNA would r i s e a f t e r a s h o r t l a g time e q u a l t o t h e time r e q u i r e d f o r assembly.  On the o t h e r hand, i f newly s y n t h e s i z e d  h i s t o n e i s n o t p r o p e r l y bound t o DNA u n t i l a c e t y l a t i o n and dea c e t y l a t i o n 12-16 hours a f t e r s y n t h e s i s would n o t appear i n l i s s u b u n i t s . s i m i l a r l y , while  (84), then  [^C]lysine  [ "*C] a c e t a t e would behave 1  [ " c ] m e t h y l l a b e l , which s h o u l d occur on 1  -122-  "old"  (subunit-bound) h i s t o n e s , would appear  immediately  in  subunits. I t may e v e n be p o s s i b l e t o s e p a r a t e of  subunits  o n v e r y weak i o n - e x c h a n g e r s ,  ences i n h i s t o n e m o d i f i c a t i o n and hence (vi) It  different  the three-dimensional  seems l i k e l y  populations  b a s e d on s m a l l charge;  s t r u c t u r e o f the chromatin  that crosslinking  studies  c o u l d be r e f i n e d  ordered  etry of Mg the  + +  i n these  the  pellets  a r r a n g e m e n t o f monomers.  t h e two s u l f h y d r y l data  f u r t h e r by X-ray d i f f r a c t i o n  a r r a y s o f monomers e . g . M g  (e.g.  groups  + +  should  pellets. give  soon.  s t u d i e s on  The s t o i c h i o m -  some i n f o r m a t i o n o n  I t may a l s o be f e a s i b l e  t o use  (one f o r e a c h H3) t o o b t a i n f u r t h e r  r e a c t i o n with mercury, a f f i n i t y  topography o f a  subunit.  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